Targeting aminoacid lipids

ABSTRACT

The present invention is directed to carrier systems comprising ether-lipids conjugated to one or more bioactive ligands and exposed on the surface of the carrier system for use in targeted delivery and/or antigen display systems. Optionally one or more further bioactive agents may be encapsulated or embedded within or attached to or adsorbed onto the carrier system. The present invention is further directed to methods of their preparation and their uses in medical applications, such as targeted delivery of bioactive agents to specific tissues or cells and antigen display systems for the study, diagnosis, and treatment of traits, diseases and conditions that respond to said bioactive agents.

FIELD OF THE INVENTION

The present invention is directed to carrier systems comprisingether-lipids conjugated to one or more bioactive ligands (and exposed onthe surface of the carrier system) for use in targeted delivery and/orantigen display systems, which carrier systems may comprise one or morefurther bioactive agents. The present invention is further directed tomethods of their preparation and their uses in medical applications,such as targeted delivery of said bioactive agents to specific tissuesor cells and antigen display systems for the study, diagnosis, andtreatment of traits, diseases and conditions that respond to saidbioactive agents.

BACKGROUND OF THE INVENTION

Molecular recognition, such as between receptor ligand,antigen-antibody, DNA-protein, sugar-lectin, RNA-ribosome, etc. is animportant principle underlying many biological systems and is beingapplied to many artificially created biological systems for use inmedical applications, such as in artificial (micro- or nano-)particulate systems including polymeric beads, vesicular lipids,microemulsions, and the like.

One important example of a molecular recognition based application isthe use of targeted delivery of diagnostic or therapeutic compounds,such as antiviral, chemotherapeutic or imaging agents, to specificsites, which allows to overcome the limitations associated withnonspecific delivery (such as in vivo clearance time, potentialtoxicity, problems associated with membrane transport of an agent andthe like) and thus greatly increases their effectiveness. Variousrecognition-based strategies have been used to improve the delivery ofcompounds into the intracellular environment (i.e. to specific cellcompartments) of a target cell to exert its biological activity, inparticular delivery through specific transporters involving the use ofbiological or artificial carriers, such as viral vectors, cationicpolymers, such as polylysine, polyarginine and the like (see, e.g. WO79/00515, WO 98/52614), lipid carriers, and various other conjugatesystems.

One widely used approach involves the use of lipid vesicles asartificial carriers, e.g. liposomes and micelles, which have beenextensively developed and analyzed as drug delivery vehicles due totheir ability to reduce systemic exposure of a bioactive agent, therebyovercoming problems associated with degradation, solubility, etc. andproviding an increase in blood circulation times. Actively targeteddelivery of a bioactive agent involves derivatizing the lipids of thelipid vesicle (either prior or after vesicle formation) with a targetingligand that serves to direct (or target) the vesicle to specific celltypes such as cancer cells or cells specific to particular tissues andorgans, such as hepatocytes, after in vivo administration (see, forexample, U.S. Pat. Nos. 6,316,024 and 6,214,388; Allen et al., Biochim.Biophys. Acta, 1237:99-108 (1995); Blume et al., Biochim. Biophys. Acta,1149:180-184 (1993)). This may be accomplished by utilizing receptorsthat are overexpressed in specific cell types, which include for examplefolic acid receptor (FR) (overexpressed in a variety of neoplastictissues, including breast, ovarian, cervical, colorectal, renal, andnasoparyngeal tumors), transferrin receptor (TfR) (overexpressed onmetastatic and drug resistant cells of most carcinomas, sarcomas andsome lymphomas and leukaemias), epidermal growth factor receptor (EGFR)(overexpressed in anaplastic thyroid cancer and breast, lung andcolorectal tumors), vascular endothelial growth factor receptor 1 and 2(VEGFR-1/2) (highly expressed on endothelial cells in tumorneovasculature), metastin receptor (overexpressed in papillary thyroidcancer), ErbB family receptor tyrosine kinases (overexpressed in asignificant subset of breast cancers), human epidermal growth factorreceptor-2 (Her2/neu) (overexpressed in breast cancers), tyrosinekinase-18-receptor (c-Kit) (overexpressed in sarcomatoid renalcarcinomas), HGF receptor c-Met (overexpressed in esophagealadenocarcinoma), CXCR4 and CCR7 (overexpressed in breast cancer),endothelin-A receptor (overexpressed in prostate cancer), peroxisomeproliferator activated receptor delta (PPAR-delta) (overexpressed inmost colorectal cancer tumors), PDGFR A (overexpressed in ovariancarcinomas), BAG-1 (overexpressed in various lung cancers), soluble typeII TGF beta receptor (overexpressed in pancreatic cancer),asialoglycoprotein receptor (overexpressed on hepatocytes), α_(v)β₃integrin receptor (overexpressed in growing tumor vascularture),legumain (a clan CD cysteine protease enriched in solid tumor tissue andoverexpressed on TAMs, tumor associated macrophages), etc.

Any agent which selectively binds to such a specific receptor cell ortissue to be treated or assayed may be attached to a lipid vesicle andact as a targeting or receptor ligand. Typically, such targeting ligandshave been attached to a lipid or lipid vesicle surface through a longchain (e.g. polymeric) linker. For example folic acid based conjugateshave been used to provide a targeted delivery approach of a therapeuticcompound useful for the treatment and/or diagnosis of a disease,allowing a reduction in the required dose of therapeutic compounds (seee.g. WO 02/094185, U.S. Pat. No. 6,335,434, WO 99/66063, U.S. Pat. No.5,416,016). Likewise, the use of galactose- and galactosamine-basedconjugates to transport exogenous compounds across cell membranes canprovide a targeted delivery approach to the treatment of liver diseasesuch as HBV and HCV infection or hepatocellular carcinoma while allowinga reduction in the required dose of therapeutic compounds required fortreatment (see e.g. U.S. Pat. No. 6,030,954, . . . ).

Another important example of a molecular recognition based aplication isthe use of antigen display systems which involve presentation of both“self” and “foreign” proteins (antigens) to the immune system togenerate T cell activation, modulation or tolerance. The receptor ligandinteractions in antigen-presenting systems that contribute to thedesired immune response or absence thereof are complex and difficult toassess, being influenced by various parameters such as ligand densities,presence of coreceptors, receptor ligand affinities and surfaceconditions. Thus a widely used approach involved using naturallyoccurring human cells (or parts thereof) whose primary function isantigen processing and presentation. But, while live cell based systemsmay be optimal for mimicking cell-cell interaction to achieve thedesired induction of tolerance or immune response, they are dependent ona regulated expression of the surface molecules including possiblyexpression of additional “costimulatory” and/or adhesion molecules onits surface membrane at a sufficient therapeutic level. Currently knownartificial systems range from genetically engineered subcellular antigenpresenting vesicles, which carry the molecules required for antigenpresentation and T-lymphocyte activation or inhibition on their surface(WO 03/039594) to systems on the basis of cell-sized, biodegradablemicrospheres based, antigen presenting system (WO 07/087341).

Clearly, there are still drawbacks to the above, molecular recognitionbased technologies and there remains a need in the art for a versatileand efficient artificial carrier system for use in molecular recognitionbased applications such as targeted delivery or antigen presentation,including simple and economic methods of their preparation.

The present application provides conjugates comprising ether-lipidshaving one or more covalently attached bioactive ligands as well asvarious carrier systems comprising these conjugates (and optionallyfurther comprising one or more bioactive agents), which allow toovercome the limitations described above.

SUMMARY OF THE INVENTION

The present invention is directed to carrier systems comprisingether-lipids conjugated with one or more bioactive ligands for use intargeted delivery and/or antigen display systems. The one or morebioactive ligands are covalently attached to the ether-lipids of generalformula I and exposed on the surface of a carrier system. Optionally atleast one bioactive agent may be encapsulated or embedded within orattached to or adsorbed onto the surface of the carrier system.

Thus, in one aspect the invention is directed to a lipidic carriersystem in form of a vesicle, such as a liposome or a micelle, comprisingat least one lipid-ligand conjugate of formula I, optionally inadmixture with further co-lipids. The at least one lipid-ligandconjugate comprises at least one ether-lipid which is covalently linkedto at least one bioactive ligand, such as an antigen ligand, a targetligand, a therapeutic ligand or a diagnostic ligand. Optionally, atleast one further bioactive agent is encapsulated or embedded in theinternal void or bilayer (membrane) or attached to or adsorbed onto thesurface of the vesicle. In some embodiments the vesicle is a liposome ora micelle.

In another aspect the invention is directed to a nanoparticulate carriersystem in form of a lipid-coated particle having an internal void or asolid core, wherein the particle is coated with at least onelipid-ligand conjugate of formula I, optionally in admixture withfurther co-lipids. The at least one lipid-ligand conjugate of formula Icomprises at least one ether-lipid which is covalently linked to atleast one bioactive ligand, such as an antigen ligand, a target ligand,a therapeutic ligand or a diagnostic ligand. In some embodiments thenanoparticulate material is a lipid-coated nanoparticle or a nanosphere.Optionally at least one further bioactive agent is encapsulated in theinternal void or embedded or dispersed in the solid core.

In another aspect the invention is also directed to the lipid-ligandconjugates themselves according to formula I, comprising an ether-lipidcharacterized by at least two ether-linked hydrocarbon chains and aheadgroup having a short, straight-chain amino acid with up to 6 carbonatoms and up to three coupling sites to which at least one bioactiveligand may be covalently attached.

The lipid-ligand conjugates relate to a compound of general formula I

whereinY represents O, N, S or a covalent bond,S₁, S₂, S₃ represent independently of each other a covalent bond or aspacer group,X₁, X₂, X₃ represent independently of each other H or a ligand groupL is a group of formula (a)

wherein the dashed line represents the linkage to N,R₁ represents H or a group of formula —(CH₂)₂—OR_(b1),represents H or a group of formula —(CH₂)₂—OR_(b2),R₂ represents H or a group of formula —CH₂—OR_(c),R_(2′) represents H or a group of formula —OR_(d) or —CH₂—OR_(d),R₃ represents H or a group of formula —(CH₂)₂—OR_(e) or —(CH₂)₃—OR_(e),R_(a), R_(b1), R_(b2), R_(c), R_(d), R_(e) represent independently ofeach other a saturated or unsaturated, straight or branched hydrocarbonchain,m is 1, 2 or 3,with the proviso that at least one of R₁, R_(1′), R₂, R_(2′), R₃ is notH and at least one of X₁, X₂, X₃ is a ligand group.

In specific embodiments the ligand group is a targeting ligand or anantigenic ligand or a therapeutic ligand or a diagnostic ligand.

Preferably, the targeting ligand is a pteroic acid derivative, a peptideand derivatives thereof, a polypeptide, a protein or a carbohydrate andthe antigenic ligand is a peptide, protein or a carbohydrate.

In a further aspect, the invention is also directed to uses of carriersystems of the invention as a drug delivery system, diagnostic system orantigen display system. Also provided are kits for preparing the carriersystems containing the lipids of the invention and pharmaceuticalformulations containing these carrier systems.

In other aspects the present invention is also directed towards methodsfor the treatment or for diagnosis of a disease comprising administeringan effective amount of a carrier system of the invention.

In yet further aspects the present invention is also directed towardsmethods for modulating an immune response comprising administering aneffective amount of a carrier system of the invention.

Other aspects of the invention include methods for transport of abiologically active compound across a membrane and/or methods ofdelivery of a biologically active compound into a cell using carriersystems of the invention

BRIEF DESCRIPTION OF THE DRAWINGS

The extent of cellular uptake for RGD decorated liposomes (obtained inExample 9) on M21 cells are evaluated on the basis of NBD-DOPE signaldetected by Guava easyCyte 8HT flowcytometer and is illustrated in FIG.1 and Table 1.

FIG. 1. Cellular uptake of an RGD targeting liposome (comprising 5%DMA-RGD) as compared to non-targeting liposome (comprising no DMA-RGD).

FIG. 2. Legumain targeting experiments of RR11a decorated liposomes (MS15-4) in comparison to control liposomes (MS 15-0) according to Example20:

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides carrier systems comprising at least oneether-lipid and at least one bioactive ligand conjugated to saidether-lipid to form a lipid-ligand conjugate of the invention. Anycarrier system which can be formed of or coated with lipid-ligandconjugates of general formula I optionally in combination with otherlipidic matrix compounds (or co-lipids) may act as a carrier systemaccording to the present invention. Typically, a carrier system of theinvention is based on a microparticulate or nanoparticulate material invarious shapes and forms, such as vesicles or spheres with an internalvoid, particles with a solid core, rods, tubes, clusters and the like.In some embodiments, a carrier system according to the invention is alipidic carrier system, such as a liposome, a micelle, wherein thelipid-ligand conjugate is forming, optionally together with other matrixlipids, the lipid wall of the vesicle. In other embodiments, a carriersystem according to the invention is a nanoparticulate carrier system,such as a nanoparticle, a nanosphere, a nanocluster, a nanotube, apolymeric bead, and the like, wherein the lipid-ligand conjugate isadsorbed, optionally together with other matrix lipids, as a coating onthe surface of the nanoparticulate carrier system. Depending on thenature and intended use of a carrier system according to the invention,one or more bioactive agents may be encapsulated or embedded within orattached to or adsorbed onto the surface of the carrier system.

As used herein, the term “bioactive” refers to an ability to elicit abiological response that is sought in a cell, tissue, system, and/orsubject (including a human being). The term “biological response” refersto the physiological reaction of a cell to a stimulus, and thus could beany cellular, neurological, chemical, inflammatory, immunologic orpathologic biological response, process or reaction by the subject. Theresponse, process or reaction can be chemical, cellular, neurological,psychological or the like. Thus, the term “bioactive ligand or bioactiveligand group” or simply “ligand” or “ligand group” as used herein refersto a ligand which elicits such a biological response and which is usedfor covalent attachment to an ether-lipid of general formula I eitherdirectly or via a spacer group (using standard chemical couplingtechniques). A bioactive ligand may be a targeting ligand, an antigenicligand, a therapeutic ligand or a diagnostic ligand.

The term “bioactive agent” or simply “agent” as used herein refers toany synthetic or naturally occurring compound (in free form, salt formor solvated or hydrated form) having a biological activity, such as atargeting agent, an antigenic agent, a therapeutic agent or a diagnosticagent, preferably a therapeutic agent or a diagnostic agent.

It is understood that the definitions of the various bioactive agentgroups and bioactive ligand groups may be overlapping.

Thus, the expression “targeting” used in conjunction with “agent” or“ligand” (for uses in targeted delivery systems) refers to a compoundwhich is capable of interacting with a complementary binding moiety at adesired location and/or under desired conditions. For example,complementary binding moieties can be ligands and anti-ligands (e.g.streptavidin and biotin, protein A or G and Fc region ofimmunoglobulins), ligands and receptors (e.g. small molecule ligands andtheir receptors, or sugar-lectin interactions), phage display-derivedpeptides, complementary nucleic acids (e.g. DNA hybrids, RNA hybrids,DNA/RNA hybrids, etc.), and aptamers. Other exemplary complementarybinding moieties include, but are not limited to, moieties exhibitingcomplementary charges, hydrophobicity, hydrogen bonding, covalentbonding, Van der Waals forces, reactive chemistries, electrostaticinteractions, magnetic interactions, etc.

A “targeting ligand” or “targeting agent” specific for a particularreceptor (a receptor agent or ligand) refers to any compound which is aspecific binding partner of a specific binding pair, wherein the otherbinding partner is a receptor. The receptor may be present attached to acell membrane or surface or in soluble form and may be presentintracellularly and/or extracellularly in a subject, preferably amammalian subject, e.g. a human or animal. Examples of a receptorinclude, without limitation, membrane receptors, soluble receptors,cloned or recombinant receptors, clan CD cysteine protease and otherproteases and other enzymes, hormone receptors, drug receptors,transmitter receptors, autocoid receptors, cytokine receptors,antibodies, antibody fragments, engineered antibodies, antibody mimics,molecular recognition units, adhesion molecules, agglutinins, integrins,and selectins. Typically, the binding affinity of a receptor ligand forits receptor may be at least 10⁻⁵M, preferably 10⁻⁷M and greater, e.g.around 10⁻⁵M to around 10⁻¹²M. Examples of a receptor agent or ligandinclude, without limitation, a peptide or polypeptide, includingderivatives thereof such as aza-peptide derivatives or derivativescontaining partially or only D-amino acids, a glycopeptide, and thelike, a protein, including a glycoprotein or phosphoprotein, acarbohydrate, glycolipid, phospholipid, oligonucleotide, polynucleotide,aptamers, spiegelmers, vitamin (e.g. vitamin B9 or folic acid, vitaminB12), antigens and fragments thereof, haptens, receptor agonists,partial agonists, mixed agonists, antagonists, drugs, chemokines,hormones (e.g. LH, FSH, TRH, TSH, ACTH, CRH, PRH, MRH, MSH, glucagon andprolactin; transferrin; lactoferrin; angiotensin; histamine; insulin;lectins), transmitters, autocoids; growth factors (for example PDGF,VEGF, EGF, TGFa, TBFß, GM-CSF, G-CSF, M-CSF, FGF, IGF, bombesins,thrombopoietin, erythropoietin, oncostatin and endothelin 1), cytokinesincluding interleukins (e.g. interleukins 1 to 15), lymphokines and cellsignal molecules, such as tumor necrosis factor (e.g. tumour necrosisfactors α and ß) and interferons (e.g. interferons α, ß and γ),prosthetic groups, coenzymes, cofactors, regulatory factors, or anyother naturally occurring or synthetic organic molecule which canspecifically bind to a receptor, including fragments, analogs and otherderivatives thereof that retain the same binding properties. The choiceof a receptor agent or ligand for use in the present invention will bedetermined by the nature of the disease, condition, or infection to beassayed and/or treated. Preferred receptor agents or ligands includevitamins (e.g. folic acid or fragments thereof), pteroic acidderivatives, peptides, including derivatives such as aza-peptidederivatives, proteins and carbohydrates. Most preferred are pteroylderivativesand peptides in particular aza-peptide derivatives.

The term “pteroyl” or “pteroic acid” as used herein represents acondensed pyrimidine heterocycle, which is linked to an aminobenzoylmoiety. As used herein a “condensed pyrimidine heterocycle” includes apyrimidine fused with a further 5- or 6-membered heterocycle, resultingin a pteridine or a pyrrolopyrimidine bicycle. Conjugation of a pteroylgroup to one or more of the reactive sites on the headgroup of an etherlipid (N- or Y-group) will result in a folate structure, wherein theheadgroup represents the glutamic acid part or a derivative thereof.Exemplary folate structures are based on a folate skeleton, i.e.pteroyl-glutamic acid resp.N-[4-[[(2-amino-1,4-dihydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamicacid, and derivatives thereof. Such folate derivatives include folateshaving optional substituents on reactive or non-reactive sites and/orwherein selected atoms have been replaced, e.g. selected heteroatoms,preferably one or two, have been replaced by carbon atoms (such as indeaza and dideaza analogs). Examples are optionally substituted folicacid, folinic acid, pteropolyglutamic acid, and folate receptor-bindingpteridines such as tetrahydropterins, dihydrofolates, tetrahydrofolates,and their deaza and dideaza analogs. Folic acid,5-methyl-(6S)-tetrahydrofolic acid and 5-formyl-(6S)-tetrahydrofolicacid are the preferred basic structures used for the compounds of thisinvention. The terms “deaza” and “dideaza” analogs refers to the artrecognized analogs having a carbon atom substituted for one or twonitrogen atoms in the naturally occurring folic acid structure. Forexample, the deaza analogs include the 1-deaza, 3-deaza, 5-deaza,8-deaza, and 10-deaza analogs. The dideaza analogs include, for example,1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs.Preferred deaza analogs compounds includeN-[4-[2-[(6R)-2-amino-1,4,5,6,7,8-hexahydro-4-oxopyrido[2,3-d]pyrimidin-6-yl]ethyl]benzoyl]-L-glutamicacid (Lometrexol) andN-[4-[1-[(2,4-diamino-6-pteridinyl)methyl]propyl]benzoyl]-L-glutamicacid (Edatrexate). In each of the above folate structure the glutamicacid portion is the portion corresponding to the headgroup of theetherlipid and thus each of the above folate structures may also includethe structures comprising the various glutamic acid derivativescorresponding to the headgroup.

The term “peptide” as used herein represents an oligopeptide consistingof 1 to 30, preferably of 2 to 20, most preferably of 3 to 10 aminoacids. Peptides are typically connected through their N-terminus,C-terminus and/or through their side chains to the reactive positions atthe head group (i.e. N- and/or Y-group) of an ether-lipid. Peptides maycontain disulfide bridges as well as ester linkages. Furthermore,peptides may bear protecting groups at the N-terminus, C-terminus and inthe side chains. The term “amino acid” includes natural occurringL-amino acids, D-amino acids, synthetic amino acids, beta amino acidsand homologues thereof.

Preferred peptides as defined above for use in the present applicationinclude e.g. cell-specific ligands such as the RGD-peptide, NGR-peptide,ATWLPPR-peptide, APRPG-peptide, SMSIARL-peptide, TAASGVRSMH-peptide,LTLRWVGLMS-peptide, CDSDSDITWDQLWDLMK-peptide, GPLPLR-peptide,HWGF-peptide, and derivatives thereof (wherein the designation of thepeptide is given in the single letter amino acid code), preferably theRGD peptide (i.e. the tripeptide amino acid sequencearginine-glycine-aspartic acid or Arg-Gly-Asp) and derivatives thereof.Derivatives of the RGD peptide include any structural modification tothe peptide including a peptide containing the RGD sequence, as well asnon-peptidic compounds comprising the RGD peptide.

The term “aza-peptide” as used herein refers to peptide analogs having anitrogen atom substituted for one or more carbon atoms in the naturallyoccurring peptide structure. Aza-peptides typically consist of 1 to 30,preferably of 2 to 20, most preferably of 3 to 10 amino acids having anitrogen atom substituted for at least one of the sp3-hybridizedcarbons, preferably for a carbon atom in alpha position of an aminoacid, most preferably for the carbon atom in alpha position of the aminoacid at the C-terminus. Aza-peptides are connected through theirN-terminus, C-terminus and/or through their side chains to the reactivepositions at the head group (i.e. N- and/or Y-group) of the ether-lipid.Aza-peptides may contain disulfide bridges as well as ester linkages.Furthermore, aza-peptides may bear protecting groups at the N-terminus,C-terminus and in the side chains. Preferred aza-peptides arederivatives of 2-azaasparagine, such as Cbz-alanylalanyl-2-azaasparagine(also known as RR11a) (Ekici et al., 2004, J. Med. Chem. 47, 1889-1892;WO 2012/031175 A9).

In other embodiments a targeting agent or ligand may also represent orcomprise at least one blocking moiety. As used herein, the term“blocking moiety” refers to moieties which mask, block, cloak, and/orsterically inhibit the activity, self-recognition, and/or self-assemblyof complementary binding moieties. For example, a blocking moiety iscapable of blocking the ability of complementary binding moieties tointeract with one another prior to a desired condition or time, when theblocking moiety is removed. A blocking moiety can include polymericentities, such as polaxamines; poloxamers; polyethylene glycol (PEG);poly(lactic-co-glycolic acid)(PLGA), peptides; synthetic polymers andthe like.

As used herein, the expression “antigen(ic)” used in conjunction with“agent” or “ligand” refers to a compound which provokes an immuneresponse against itself or portions thereof. The term “immune response”refers to recognition of an antigen or parts thereof by an immuneeffector cell. This includes T cell mediated and/or B cell mediatedimmune responses that are influenced by modulation of T cellco-stimulation. The term immune response further includes immuneresponses that are indirectly effected by T cell activation such asantibody production (humoral responses) and the activation of otherimmune effector cells including, but not limited to, monocytes,macrophages, NK cells and cytotoxic T lymphocytes (CTLs), for exampleCTL lines, CTL clones, and CTLs from tumor, inflammatory, or otherinfiltrates. Certain diseased tissue express specific antigens and CTLsspecific for these antigens have been identified. For example,approximately 80% of melanomas express the antigen known as gp-100. Oneof the most effective and desirable procedures to prevent microbialinfections and pathogenic processes and thus combat such diseases arevaccines, which cause a stimulation of an immune response in a hostorganism prior to an actual infection or onset of a disease byintroducing antigens or immunogens into the host organism.

A skilled person will understand that any macromolecule, includingvirtually any biological molecule (proteins, peptides, lipids,lipoproteins, glycans, glycoproteins, nucleic acids derivatives, such asoligonucleotides, polynucleotides, genomic or recombinant DNA) may serveas an antigen. An antigen may be synthesized chemically or biologically,or may be derived from recombinant or genomic DNA or can be derived froma biological sample, such as a tissue sample, a tumor sample, a cell ora biological fluid. Antigens may include, but are not limited to, viralantigens, bacterial antigens, fungal antigens, protozoal and otherparasitic antigens, tumor antigens, antigens involved in autoimmunedisease, addiction, allergy and graft rejection, and other miscellaneousantigens. Representative examples of an antigen may be a protein orpeptide of bacterial, fungal, protozoan, or viral origin, or a fragmentderived from these antigens, which include, but are not limited to,Streptococcus species, Candida species, Brucella species, Salmonellaspecies, Shigella species, Pseudomonas species, Bordetella species,Clostridium species, Norwalkvirus, Bacillus anthracis, Mycobacteriumtuberculosis, human immunodeficiency virus (UV), Chlamydia species,human Papillomaviruses, Influenza virus, Paramyxovirus species, Herpesvirus, Cytomegalovirus, Varicella-Zoster virus, Epstein-Barr virus,Hepatitis viruses, Plasmodium species, Trichomonas species, sexuallytransmitted disease agents, viral encephalitis agents, protozoan diseaseagents, fungal disease agents, bacterial disease agents, cancer cells,or mixtures thereof.

Immunization of a subject may be enhanced by the use of multiple copiesof an antigen as a multivalent display and is desirable in case ofantigen ligands such as small peptides or carbohydrates, that aredifficult to administer and generally fail to elicit an effective immuneresponse due to the hapten-related size issues. Thus, as used herein,the term “multivalent” refers to the display of more than one copy ortype of antigen on a carrier system.

The term “antigen-presenting system” or “antigen display system” as usedherein refers to a naturally occurring or synthetic system, which (i)can present at least one antigen (or part thereof) in such a way thatthe at least one antigen (or part thereof) can be recognized or bound byan immune effector molecule, e.g. a T-cell antigen receptor on thesurface of a T cell, or (ii) is capable of presenting at least oneantigen (or part thereof) in the form of an antigen-MHC complexrecognizable by specific effector cells of the immune system, andthereby inducing an effective cellular immune response against theantigen (or part thereof) being presented. In the context of the presentinvention, the term “recognized” refers to (i) a lipid compoundconjugated to at least one antigenic ligand (or a composition orformulation thereof) which is recognized and bound by an immune effectorcell wherein such binding is sufficient to initiate an effective immuneresponse, or to (ii) a lipid compound conjugated to at least onetargeting ligand (or a composition or formulation thereof) which isrecognized and bound by its corresponding receptor or to a combinationof both (a) and (b). Assays for determining whether a targeting or anantigenic ligand is recognized by a receptor or an immune effector cell,respectively, are known in the art and are described herein.

As used herein, the expression “therapeutic” used in conjunction with“agent” or “ligand” refers to a compound which is capable of exerting abiological effect in vitro and/or in vivo that is therapeutic in nature.A therapeutic ligand may be neutral or positively or negatively charged.Examples of suitable bioactive agents include pharmaceuticals and drugs,synthetic organic molecules, proteins, vitamins, steroids, siRNA, miRNA,adjuvants and genetic material.

The term “genetic material” refers generally to nucleosides,nucleotides, and polynucleotides, including deoxyribonucleic acid (DNA)and ribonucleic acid (RNA). The genetic material may be made bysynthetic chemical methodology known to one of ordinary skill in theart, or by the use of recombinant technology, or by a combination of thetwo. The DNA and RNA may optionally comprise unnatural nucleotides andmay be single or double stranded. “Genetic material” refers also tosense and anti-sense DNA and RNA, that is, a nucleotide sequence whichis complementary to a specific sequence of nucleotides in DNA and/orRNA.

The term “pharmaceutical” or “drug” refers to any therapeutic orprophylactic agent which is used in the prevention, diagnosis,alleviation, treatment or cure of a disease or injury in a patient. Itis understood that the bioactive agents to be entrapped or embedded inthe lipid compositions or attached to or adsorbed onto the surface ofthe lipid compositions of the invention are not restricted to anyparticular class of biologically active material in terms ofphysicochemical properties, molecular size or the source of origin, i.e.synthetic, biotechnological materials, etc. Thus the pharmaceutical maybe, for example, chosen from any of the following therapeutic class:analgesic, anesthetic, anti-Alzheimer's, anti-asthma agent,anti-Parkinsonism, antiallergic, antianginal, antiarrhythmic,antiarthritic, antiasthmatic, antibacterial, antibiotic, anticancer,anticoagulant, antidepressant, antidiabetic, antiemetic, antiepileptic,antifungal, antiglaucoma, anti-gout, antihistamine,antihyperprolactinemia, antihypertensive, antiinflammatory,antimigraine, anti-neoplastic, antiobesity, antiparasitic,anti-protozoal, anti-phyretics, antipsoriatic, antipsychotic,antithrombotic, antiulcer, antiviral, anxiolytic, benign prostatichypertrophy, bronchodilator, calcium metabolism, cardiotonic,cardiovascular agent, chelator AND antidote, chemopreventive agent,contraception, diuretic, dopaminergic agent, gastrointestinal agent,gastroprokinetic, hematopoiesis, hemophilia, hormone, hormonereplacement therapy, hypnotic, hypocholesterolemic, hypolipidemic,immunomodulator, immunostimulant, immunosuppressant, lipid regulatingagent, male sexual dysfunction, multiple sclerosis, muscle relaxant,neuroleptic, nootropic, osteoporosis, phytoestrogen, plateletaggregation inhibitor, prostaglandin, radioenhencer for radiotherapy,relaxant and stimulant, respiratory distress syndrome, urinaryincontinence, vasodilator, vitamin/nutritional, vulnerary and xanthine.Active agents belonging to these classes can be used in the previouslymentioned compositions.

As used herein, the expression “diagnostic” used in conjunction with“agent” or “ligand” refers to a compound which is capable of diagnosingthe presence or absence of a disease in a patient. The diagnostic agentsmay be neutral or positively or negatively charged. Examples of suitablediagnostic agents include, synthetic organic molecules and heavy metalcomplexes, such as contrast agents for use in connection with magneticresonance imaging, ultrasound or computed tomography of a patient.

The choice of a targeting or antigenic or therapeutic or diagnosticligand or agent for use with the carrier systems of the presentinvention will be determined by the nature of the disease, condition, orinfection to be assayed and/or treated.

These and more aspects of the invention are disclosed in the followingparagraphs.

A. Lipid-Ligand Conjugates

The term “lipid-ligand conjugate” as used herein refers to a compound ofthe invention, which comprise a linear, bifunctional amino acid at thehead group, more specifically a 2-amino-alkanedioic acid (having up tosix carbon atoms), such as aspartic acid, glutamic acid, etc., and whichare conjugated at coupling sites of the head group to one or morebioactive ligands to form a “lipid-ligand conjugate”. The term“ether-lipid (compound)” or “lipid (compound)” as used herein refers tothe precursor, i.e. the corresponding lipid prior to conjugation to oneor more bioactive ligands.

Thus in one aspect the invention is directed towards lipid-ligandconjugates according to formula I

whereinY represents O, N, S or a covalent bond,S₁, S₂, S₃ represent independently of each other a covalent bond or aspacer group,X₁, X₂, X₃ represent independently of each other H or a ligand group,L is a group of formula (a)

wherein the dashed line represents the linkage to N,R₁ represents H or a group of formula —(CH₂)₂—OR_(b1),R_(1′) represents H or a group of formula —(CH₂)₂—OR_(b2),R₂ represents H or a group of formula —CH₂—OR_(c),R_(2′) represents H or a group of formula —OR_(d) or —CH₂—OR_(d),R₃ represents H or a group of formula —(CH₂)₂—OR_(e) or —(CH₂)₃—OR_(e),R_(a), R_(b1), R_(b2), R_(c), R_(d), R_(e) represent independently ofeach other a saturated or unsaturated, straight or branched hydrocarbonchain,m is 1, 2 or 3,with the proviso that at least one of R₁, R_(1′), R₂, R_(2′), R₃ is notH and at least one of X₁, X₂, X₃ is a ligand group.

As used herein, the terms “conjugated” (or “conjugation”), “linked”,“attached”, when used with respect to two or more moieties, refers tophysical association of two or more moieties by covalent bonds (eitherdirectly or through a spacer).

The corresponding (ether-)lipid compounds which include non-derivatized(lipid) compounds, wherein the headgroup (i.e. the N- and Y-group) donot carry a ligand group but are in free form, in protected form or inactivated form), as well as derivatized (lipid) compounds, wherein theheadgroup (i.e. the N- and Y-group) is derivatized with one or morespacer groups, are part of an application filed concurrently, which isincorporated herein in its entirety.

In a first embodiment of a compound of I, group R₃ is H. Morespecifically, either (i) R₃ is H and R₁ and R_(1′) are H, or (ii) R₃ isH and R₂ and R_(2′) are H. Thus, in this first embodiment the inventionis directed towards compounds of formula Ia,

wherein L is a group of formula (a)

and wherein S₁, S₂, S₃, X₁, X₂, X₃, Y, R₁, R_(1′), R₂, R_(2′), R_(a),and m are defined as above for a compound of formula I.

More specifically, the invention is directed towards compounds offormula Ia, wherein L is a group of formulas (b) or (c)

wherein R₁, R₁, R₂, R_(2′), R_(a) are defined as above,with the proviso that in formula (b) one of R₂ and R_(2′) is not H, andin formula (c) one of R₁ and R_(1′) is not H, and at least one of X₁,X₂, X₃ is a ligand group.

In one preferred embodiment of group (b) R₂ is H and R_(2′) is —OR_(d)or —CH₂—OR_(d). In another preferred embodiment of group (b) R₂ is—CH₂—OR_(c) and R_(2′) is —OR_(d) or R_(2′) is —CH₂—OR_(d).

Thus, the invention is preferably directed to compounds wherein L is agroup of formula (b1), (b2), (b3) or (b4):

wherein S₁, S₂, S₃, X₁, X₂, X₃, Y, m, R_(a), R_(c), R_(d) are defined asabove.

In one preferred embodiment of group (c), one of R₁ and R_(1′) is H. Inanother preferred embodiment of group (c) neither of R₁ and R_(1′) is H.

Thus, the invention is preferably also directed to compounds wherein Lis a group of formula (c1) or (c2):

wherein S₁, S₂, S₃, X₁, X₂, X₃, Y, m, R_(a), R_(b1), R_(b2) are definedas above.

In a second embodiment, R₁, R_(1′), R₂, R_(2′) are H and R₃ is either agroup of formula —(CH₂)₂—OR_(e) or —(CH₂)₃—OR_(e).

Thus, in this second embodiment the invention is directed towardscompounds of formula Ib,

wherein R₃ is a group of formula —(CH₂)₂—OR_(e) or —(CH₂)₃—OR_(e), andS₁, S₂, S₃, X₁, X₂, X₃, Y, R_(a), R_(e) and m are defined as above.

Most preferred embodiments of the invention are thus compounds offormula I, which are compounds of formulas II or III

whereinY represents O, N, S or a covalent bond,S₁, S₂, S₃ represent independently of each other a covalent bond or aspacer group,X₁, X₂, X₃ represent independently of each other H or a ligand group,R₁ represents H or a group of formula —(CH₂)₂—OR_(b1),R_(1′) represents H or a group of formula —(CH₂)₂—OR_(b2),R₂ represents H or a group of formula —CH₂—OR_(c),R_(2′) represents H or a group of formula —OR_(d) or —CH₂—OR_(d),R_(a), R_(b1), R_(b2), R_(c), R_(d) represent independently of eachother a saturated or unsaturated, straight or branched hydrocarbonchain,m is 1, 2 or 3,with the proviso that (i) in formula II one of R₂ and R_(2′) is not H,and in formula III one of R₁ and R_(1′) is not H, and that (ii) at leastone of X₁, X₂, X₃ is a ligand group.

More specific embodiments of compounds of formula II are compounds offormula IIa, IIb, IIc or IId,

wherein S₁, S₂, S₃, X₁, X₂, X₃, Y, R_(a), R_(c), R_(d) and m are definedas above for a compound of formula II.

More specific embodiments of compounds of formula III are compounds offormula IIIa or IIIb,

wherein S₁, S₂, S₃, X₁, X₂, X₃, Y, R_(a), R_(b1), R_(b2) and m aredefined as above for a compound of formula III.

Other most preferred embodiments of compounds of formula I are compoundsof formulas IVa and IVb,

whereinY represents O, N, S or a covalent bond,S₁, S₂, S₃ represent independently of each other a covalent bond or aspacer group,X₁, X₂, X₃ represent independently of each other H or a ligand group,R_(a), R_(e) represent independently of each other a saturated orunsaturated, straight or branched hydrocarbon chain, andm is 1, 2 or 3,with the proviso that at least one of X₁, X₂, X₃ is a ligand group.

A person skilled in the art will appreciate that the ligand-lipid of thepresent invention (or compounds of the present invention) contain one ormore chiral centers and/or double bonds and therefore, may exist asstereoisomers, such as double-bond isomers (i.e., geometric isomers,e.g. Z/E isomers or cis/trans isomers), enantiomers or diastereomers.Accordingly, when stereochemistry at chiral centers is not specified,the chemical structures depicted herein encompass all possibleconfigurations at those chiral centers including the stereoisomericallypure form (e.g., geometrically pure, enantiomerically pure ordiastereomerically pure) the enriched form (e.g., geometricallyenriched, enantiomerically enriched or diastereomerically enriched) andenantiomeric and stereoisomeric mixtures. The individual isomers may beobtained using the corresponding isomeric forms of the startingmaterial. Alternatively, enantiomeric and stereoisomeric mixtures can beresolved into their component enantiomers or stereoisomers usingseparation techniques or chiral synthesis techniques well known to theskilled artisan. The compounds of the invention described herein mayalso exist in several tautomeric forms including the enol form, the ketoform and mixtures thereof. Accordingly, the structures depicted hereinencompass all possible tautomeric forms of the illustrated compounds.

The term “saturated or unsaturated, straight or branched hydrocarbonchain” as used herein refers to a saturated or unsaturated, straight orbranched hydrocarbon chain having 6 to 30, preferably 10 to 22 carbonatoms.

The term “saturated” in combination with hydrocarbon chain refers to astraight or branched alkyl chain, containing 6 to 30, preferably 10 to22 carbon atoms. Examples include, but are not limited to, capryl(decyl), undecyl, lauryl (dodedecyl), myristyl (tetradecyl), cetyl(hexadecyl), stearyl (octadecyl), nonadecyl, arachidyl (eicosyl),heneicosyl, behenyl (docosyl), tricosyl, tetracosyl, pentacosyl,including branched isomers thereof, e.g. isolauryl, anteisolauryl,isomyristyl, anteisomyristyl, isopalmityl, anteisopalmityl, isostearyl,anteisostearyl or phytanyl (3,7,11,15-tetramethyl-hexadecanyl).

The term “unsaturated” in combination with hydrocarbon chain indicatesthat fewer than the maximum possible number of hydrogen atoms are bondedto each carbon in the chain giving rise to one or more carbon-carbondouble or triple bonds. In preferred embodiments, the number ofunsaturated bond(s) in an unsaturated hydrocarbon chain is 1, 2, 3 or 4,preferably 1 or 2.

Examples of alkenyl groups include, but are not limited to,monounsaturated alkenyls, such as decenyl, undecenyl, dodecenyl,palmitoleyl, heptadecenyl, octadecenyl (elaidyl, oleyl, ricinolenyl),nonadecenyl, eicosenyl, heneicosenyl, docosenyl (erucyl), tricosenyl,tetracosenyl, pentacosenyl, and the branched chain isomers thereof, aswell as polyunsaturated alkenyls such as octadec-9,12-dienyl (linoleyl,elaidolinoleyl), octadec-9,12,15-trienyl (linolenyl, elaidolinolenyl),9(Z),11(E),13(E)-octadecatrienyl (eleostearyl), andeicos-5,8,II,14-tetraenyl.

Examples of alkynyl groups include, but are not limited tohexadec-7-ynyl and octadec-9-ynyl.

The term “branched” in combination with hydrocarbon refers to ahydrocarbon chain having a linear series of carbon atoms as a main chainwith at least one substituent of one or more carbon atoms as subordinatechain (or branching groups). Examples of subordinate chains include oneor more (C1-6)alkyl groups, such as methyl, ethyl, propyl, isopropyl,n-butyl, sec-butyl group, tert-butyl, pentyl, hexyl and the like, one ormore (C1-6)alkenyl groups, such as vinyl, allyl, propenyl, isopropenyl,2-butenyl and the like, or one or more (C1-6)alkynyl groups, such asethynyl, propynyl, butynyl and the like. Preferred subordinate chainsare (C1-6)alkyl groups, most preferred methyl and ethyl.

The compounds of the invention comprise preferably at least twohydrocarbon chains, preferably 2, 3, 4, 5 or 6 hydrocarbon chains, mostpreferably 2 or 3 hydrocarbon chains, wherein the main chain of thehydrocarbon chains are the same or different, preferably the same, andare selected from an alkyl chain, an alkenyl chain, and an alkynylchain, preferably an alkyl and an alkenyl chain. In one preferredembodiment, the compounds of the invention carry two alkyl chains, whichcan be the same or different, preferably the same.

In a specific embodiment of a compound of the invention the hydrocarbonchains R_(a), R_(b1), R_(b2), R_(c), R_(d), R_(e) are preferablyselected from myristyl, palmityl, stearyl, oleyl, linoleyl andphytanoyl.

The terms “alkyl”, “alkoxy”, “alkenyl”, “alkynyl” as used herein havethe following meanings:

The term “alkyl refers to a straight or branched alkylchain, containing1 to 12, preferably 1 to 8 carbon atoms. Examples of alkyl groupsinclude, but are not limited to, methyl, ethyl, n-propyl, i-propyl,n-butyl, i-butyl, and t-butyl. The term “alkoxy” refers to an —O-alkylradical. Examples of alkoxy groups include, but are not limited to,methoxy, ethoxy, and butoxy. The term “alkenyl” refers to a straight orbranched unsaturated alkyl group having one or more carbon-carbon doublebonds. The above alkyl, alkenyl, and alkoxy groups may be optionallysubstituted with further groups. Examples of substituents include, butare not limited to, halo, hydroxyl, amino, cyano, nitro, mercapto,alkoxycarbonyl, amido, carboxy, alkylsulfonyl, alkylcarbonyl, carbamido,carbamyl, carboxyl, thioureido, thiocyanato, sulfonamido, aryl,heteroaryl, cyclyl, and heterocyclyl. The term “aryl” refers to anaromatic carbocyclic radical containing about 6 to about 10, preferably5 to 7 carbon atoms. The aryl group may be optionally substituted withone or more aryl group substituents which may be the same or different,where “aryl group substituent” includes alkyl, alkenyl, alkynyl, aryl,aralkyl, hydroxy, alkoxy, aryloxy, aralkoxy, carboxy, aroyl, halo,nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl,aralkoxycarbonyl, acyloxy, acylamino, aroylamino, carbamoyl,alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene and—NRR′, wherein R and R′ are each independently hydrogen, alkyl, aryl andaralkyl. Exemplary aryl groups include substituted or unsubstitutedphenyl, naphthyl, pyrenyl, anthryl, and phenanthryl.

The term “heteroaryl” refers to an aryl moiety as defined above havingat least one heteroatom (e.g., N, O, or S). Examples of a heteroarylmoiety include furyl, furylene, fluorenyl, pyrrolyl, thienyl, oxazolyl,imidazolyl, thiazolyl, pyridyl, pyrimidinyl, quinazolinyl, quinolyl,isoquinolyl and indolyl. The term “(hetero)aryloxy” refers to an(hetero)aryl-O-group wherein the (hetero)aryl group is as previouslydescribed. Exemplary aryloxy groups include phenoxy and naphthoxy. Theterm “(hetero)aralkyl” refers to an (hetero)aryl-alkyl-group wherein(hetero)aryl and alkyl are as previously described. Exemplary aralkylgroups include benzyl, phenylethyl and naphthylmethyl. The term“(hetero)aralkyloxy” refers to an (hetero)aralkyl-O-group wherein the(hetero)aralkyl group is as previously described. An exemplaryaralkyloxy group is benzyloxy.

The term “cycloalkyl” refers to a saturated or unsaturated,non-aromatic, cyclic hydrocarbon moiety having 6 to 10 carbon atoms,such as cyclohexyl or cyclohexen-3-yl. The term “heterocycloalkyl”refers to a cycloalkyl as defined herein having at least one ringheteroatom (e.g., N, O, or S), such as 4-tetrahydropyranyl or 4-pyranyl.

Aryl, heteroaryl, cycloalkyl, heterocycloalkyl as mentioned hereininclude both substituted and unsubstituted moieties, unless specifiedotherwise. Possible substituents on cycloalkyl, heterocycloalkyl, aryl,and heteroaryl include (C1-C10)alkyl, (C2-C10)alkenyl, (C2-C10)alkynyl,(C3-C8)cycloalkyl, (C5-C8)cycloalkenyl, (C1-C10)alkoxy, aryl, aryloxy,heteroaryl, heteroaryloxy, amino, (C1-C10)alkylamino,(C1-C20)dialkylamino, arylamino, diarylamino, hydroxyl, halogen, thio,(C1-C10)alkylthio, arylthio, (C1-C10)alkylsulfonyl, arylsulfonyl,acylamino, aminoacyl, amidino, guanidine, ureido, cyano, nitro, acyl,acyloxy, carboxyl, and carboxylic ester. Cycloalkyl, heterocycloalkyl,aryl, and heteroaryl can also be fused with each other.

Group Y is O, N, S or a covalent linkage, preferably 0 or N, mostpreferably N. It is understood that if group Y is a covalent linkage,—S₁—X₁ is directly linked to the CO-group.

The term “spacer” or “spacer group” (or groups S₁, S₂, S₃) as usedherein refers to a bivalent branched or unbranched chemical group whichallows to link an ether-lipid of the invention to one or more bioactiveligands X₁, X₂, X₃ in sufficient distance to eliminate any undesiredinteraction between ether-lipid and ligand and/or to reduce any sterichindrance (caused by the ether-lipid itself or any other neighbouringmolecules) that may impact the biological activity of the ligand (suchas affinity binding of ligands to their target). Depending on theintended use of a conjugate of ether-lipid and bioactive ligand, thespacer groups may be of different length and may be (hydrolytically,enzymatically and chemically) stable or may include a cleavable linkage.Cleavable linkages of the invention may be selected to be cleaved viaany form of cleavable chemistry, e.g. chemical, enzymatic, hydrolyticand the like. Exemplary cleavable linkers include, but are not limitedto, protease cleavable peptide linkers, nuclease sensitive nucleic acidlinkers, lipase sensitive lipid linkers, glycosidase sensitivecarbohydrate linkers, pH sensitive linkers, hypoxia sensitive linkers,photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers,ultrasound-sensitive linkers, x-ray cleavable linkers, etc.

It is understood that the spacers may or may not be end-group activatedto allow for linkage of the spacer modified compound of the invention toa further moiety, such as bioactive group.

In specific embodiments, a “spacer group” (or groups S₁, S₂, S₃)represents a short spacer group or a long-chain spacer group selectedfrom an alkylene chain optionally comprising one or more of the groupsselected from ketone, ester, ether, amino, amide, amidine, imide,carbamate or thiocarbamate functions, glycerol, urea, thiourea, doublebonds or aromatic rings.

More specifically, a short spacer group (or groups S₁, S₂, S₃) may bechosen from (C1-C12)alkyl, (C2-C12)alkenyl, aryl, aralkyl, heteroaryl. Along-chain spacer group (or groups S₁, S₂, S₃) may be chosen frompolymeric radicals of formula —W—(CH₂—)_(k)—W′—, wherein k is an integerbetween 13 and 3000, and W and W′ are reactive groups able to react withamino, carboxyl, hydroxy or thio groups and wherein one or more of thenon-adjacent CH₂ groups may independently be replaced by aryl,heteroaryl, —CH═CH—, —C≡C—, or a hydrophilic (or polar) group selectedfrom —O—, —CO—, —CO—O—, —O—CO—, —NR′—, —NR′—CO—, —CO—NR′—, —NR′—CO—O—,—O—CO—NR′—, —NR′—CO—NR′—, and —O—CO—O—, wherein R′ represents hydrogenor (C1-C12)alkyl. It is understood that replacing more than onenon-adjacent CH₂ group by the same group may yield in polymeric chainhaving a specific repeating unit (e.g. a polyester, polyether,polyimide, etc).

Preferred spacer groups include hydrophilic polymeric radicals (with anincreased affinity for aqueous solutions), i.e. polymers containingrepeating structural units that comprise one or more of the abovehydrophilic (or polar) groups in their alkylene backbone. Typicalexamples of hydrophilic polymeric radicals includepolyoxy(C₂-C₃)alkylenes (e.g. polyethylene glycol (PEG) or polypropyleneglycol (PPG)), polysaccharides (e.g. dextran, pullulan, chitosan,hyaluronic acid), polyamides (e.g. polyamino acids, semisyntheticpeptides and polynucleotides); polysialic acid, polyesters (e.g.polylactide (PLA), polylactid-co-glycolid (PLGA)), polycarbonates,polyethyleneimines (PEI), polyimides polyvinyl acetate (PVA).

A preferred spacer is “PEG” or “polyethylene glycol”, which encompassesany water-soluble poly(ethylene oxide). Typically, “PEG” means a polymerthat contains a majority, e.g. >50%, of subunits that are —CH₂CH₂O—.Different forms of PEG may differ in molecular weights, structures orgeometries (e.g., branched, linear, forked PEGs, multifunctional, andthe like). PEGs for use in the present invention may preferably compriseone of the two following structures: “—O(CH₂CH₂O)_(m)—” or“—CH₂CH₂O(CH₂CH₂O)_(m)—CH₂CH₂—,” where m is 3 to 3000, and the terminalgroups and architecture of the overall PEG may vary. As indicated above,depending on its use, PEG may be in end-capped form.

When PEG is defined as “—O(CH₂CH₂O)_(m)—” the end capping group isgenerally a carbon-containing group typically comprised of 1-20 carbonsand is preferably alkyl (e.g., methyl, ethyl or benzyl) althoughsaturated and unsaturated forms thereof, as well as aryl, heteroaryl,cyclyl, heterocyclyl, and substituted forms of any of the foregoing arealso envisioned. When PEG is defined as “—CH₂CH₂O(CH₂CH₂O)_(m)—CH₂CH₂—”,the end capping group is generally a carbon-containing group typicallycomprised of 1-20 carbon atoms and an oxygen atom that is covalentlybonded to the group and is available for covalently bonding to oneterminus of the PEG. In this case, the group is typically alkoxy (e.g.,methoxy, ethoxy or benzyloxy) and with respect to the carbon-containinggroup can optionally be saturated and unsaturated, as well as aryl,heteroaryl, cyclyl, heterocyclyl, and substituted forms of any of theforegoing. The other (“non-end-capped”) terminus is typically ahydroxyl, amine or an activated group that can be subjected to furtherchemical modification when PEG is defined as“—CH₂CH₂O(CH₂CH₂O)_(m)—CH₂CH₂—” In addition, the end-capping group canalso be a silane.

A review for the preparation of various end-group functionalized oractivated PEG is known in the art (see for example Zalipsky S.,Bioconjug. Chem., 6, 150-165 (1995)).

Methods for conjugating a bioactive ligand (X₁ and/or X₂ and/or X₃) toan ether-lipid (i.e. compounds of formula I wherein X₁, X₂, X₃ are H)include covalent binding of one or more bioactive ligands X₁, X₂, X₃ toone or more of the reactive positions at the head group (i.e. N- and/orY-group) of one or more individual ether-lipid. Thus, one bioactiveligand may be attached to one or more sites of one individualether-lipid or to more than one site of more than one individualether-lipid. Alternatively, two or three bioactive ligands are attachedto the coupling sites of one individual ether-lipid. The one or morebioactive groups maybe attached directly to the ether-lipid or via aspacer group.

Typically, methods for linking may generally include the steps of:

a) providing a lipid compound of formula I, wherein X₁, X₂, X₃ are H,carrying one or more coupling sites on one or more of groups S₁, S₂, S₃,

b) providing an antigen ligand carrying a reactive group suitable forreacting with the one or more coupling sites, and

c) reacting the lipid compound with the antigen to obtain a ligand-lipidconjugate.

The term “coupling site” or “coupling group”, as used herein, refers toa reactive or functional group capable of reacting with a correspondingreactive or functional group (or two coupling partners) in a couplingreaction to form a covalent bond (C—C, C—O, C—N, C—S-linkage). Thechoice of conjugation (or coupling) method depends on various factors,such as the nature of the bioactive ligand to be attached, i.e. physicalattributes (e.g. size, charge, etc.), the nature of the reactive groupspresent on the bioactive ligand, and the like.

In some embodiments, conjugation is carried out in the presence of abifunctional agent (i.e., an agent with two functional (end)groups),preferably a heterobifunctional agent (i.e., an agent with two differentfunctional (end)groups). The use of such a (hetero)bifunctional agentresults in a lipid-ligand conjugate wherein lipid and ligand may bedirectly linked to each other or separated by a spacer. Typicalfunctional groups include, but are not limited to, groups such assuccinimidyl esters, maleimides, and pyridyldisulfides. In someembodiments, the bifunctional agent is selected from, but not limitedto, e.g., carbodiimides, N-hydroxysuccinimidyl-4-azidosalicylic acid(NHS-ASA), dimethyl pimelimidate dihydrochloride (DMP),dimethylsuberimidate (DMS), 3,3′-dithiobispropionimidate (DTBP),N-Succinimidyl 3-[2-pyridyldithio]-propionamido (SPDP), succimidylα-methylbutanoate, biotinamidohexanoyl-6-amino-hexanoic acidN-hydroxy-succinimide ester (SMCC),succinimidyl-[(N-maleimidopropionamido)-dodecaethyleneglycol]ester (NHS-PEO12), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB),N-succinimidyl S-acetylthioacetate (SATA),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) andN-□-maleimidobutyryloxy-succinimide ester (GMBS), succinmidyl dicarbonylpentane or disuccinimidyl suberate. In other embodiments, thebifunctional agent is Traut's Reagent 2-iminothiolane in combinationwith SPDP. In still a further embodiment the linker is. In a furtherembodiment, the bifunctional agent is selected among those disclosed inThe Pierce Products Catalogue (Pierce Chemical Company, USA) and theDouble Agents™ Cross-Linking Reagents Selection Guide (Pierce ChemicalCompany), which are herein incorporated by reference.

Preferred conjugation methods include carbodiimide-mediated amideformation and active ester maleimide-mediated amine and sulfhydrylcoupling, and the like.

For example, a thiol-containing molecule may be reacted with anamine-containing molecule using a heterobifunctional cross-linkingreagent, e.g., a reagent containing both a succinimidyl ester and eithera maleimide, a pyridyldisulfide, or an iodoacetamide. Amine-carboxylicacid and thiol-carboxylic acid cross-linking, maleimide-sulfhydrylcoupling chemistries (e.g., the maleimidobenzoyl-N-hydroxysuccinimideester (MBS) method), etc., may be used.

Polypeptides can conveniently be conjugated to an etherlipid via amineor thiol groups in lysine or cysteine side chains respectively, or by anN-terminal amino group. Likewise, oligonucleotides can conveniently beconjugated to an etherlipid through a unique reactive group on the 3′ or5′ end, e.g. a sulfhydryl, amino, phosphate group or the like. Reactivesulfhydryl groups may be coupled to a lipid of formula I having a freeamino group (e.g. groups N and Y) through the use of reagents such as(i) N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and long chainSPDP (lc-SPDP) yielding a cleavable disulfide bond between the lipid andthe oligonucleotide or polypeptide, or (ii) succinimidyl-iodoacetate toproduce non-cleavable bonds between the lipid and oligonucleotide orpolypeptide. These and other conjugation techniques are known in the art(see e.g. U.S. Pat. No. 5,512,439; WO 01/22995; Greg Hernanson“Bioconjugate Techniques,” Academic Press, 1996; Gordon Bickerstaff“Immobilization of Enzymes and Cells,” Humana Press, 1997).

A skilled person will know which functional group or functional groups(e.g., amine, carbonyl or carboxyl groups on the spacer group S₁, S₂, S₃of the headgroup of an ether-lipid of formula I to choose to allowconjugation to occur with a bioactive ligand according to the abovedescribed conjugation methods.

Additional general information on conjugation methods can be found e.g.in “Cross-Linking,” Pierce Chemical Technical Library, available at thePierce web site and originally published in the 1994-95 Pierce Catalog,and references cited therein; Wong SS, Chemistry of Protein Conjugationand Cross-linking, CRC Press Publishers, Boca Raton, 1991; andHermanson, G. T., Bioconjugate Techniques, Academic Press, Inc., SanDiego, 1996.

Molar ratios to be used in conjugating one or more ligands to anether-lipid compound of formula I may be readily optimized by a skilledperson. Typically, it may range from about 1:1 to about 10:1 lipidcompound to ligand.

In the general method presented above, any suitable method may be usedto purify an intermediate conjugated compound, such as by preparativereverse phase HPLC (RP-HPLC), by membrane filtration, such asultrafiltration or diafiltration. Unreacted reactants may be removed bysize exclusion chromatography, such as gel filtration, or equilibriumdialysis. The final conjugate may also be purified using any suitablemeans, including for instance gel filtration, membrane filtration, suchas ultrafiltration, or ion exchange chromatography, or a combinationthereof. The lipid-ligand conjugates of the invention are particularlysuitable for use in the preparation of lipidic or nanoparticulatecarrier systems, such as liposomes, micelles and nanoparticles.

B. Lipidic Carrier Systems

In a further aspect the invention is directed to a lipidic carriersystem comprising one or more lipid-ligand conjugates of the inventionoptionally in combination with other co-lipids.

Exemplary lipidic carrier systems preferably include lipid(ic) vesicles.The term “lipid(ic) vesicle” (or present vesicles or vesicles of theinvention) is used interchangeably with the expression lipidic carriersystems and refers to a spherical entity which is characterized by thepresence of an internal void. Typically, vesicles of the invention areformed from one or more lipid-ligand conjugates optionally incombination with other synthetic or naturally-occurring lipids(co-lipids). In any given vesicle of the invention, the lipids may be inthe form of a monolayer or a bilayer. In the case of more than one mono-or bilayer, the mono- or bilayers are generally concentric. The presentvesicles include such entities commonly referred to as liposomes (i.e. avesicle including one or more concentrically ordered lipid bilayer(s)with an internal void), micelles (i.e. a vesicle including a singlelipid monolayer with an internal void), and the like. Thus, the lipidsmay be used to form a unilamellar vesicle (comprised of one monolayer orbilayer), an oligolamellar vesicle (comprised of about two or aboutthree monolayers or bilayers) or a multilamellar vesicle (comprised ofmore than about three monolayers or bilayers).

The internal void of the vesicles are generally filled with a liquid,including, for example, an aqueous liquid, a gas, a gaseous precursor,and/or a solid material, including, for example, one or more bioactiveagents, see also hereinafter.

In some embodiments the ligand of the lipid-ligand conjugate is atargeting ligand (to yield a targeted lipidic carrier system or targetedliposome). In other embodiments the ligand of the lipid-ligand conjugateis an antigenic ligand (to yield an antigenic lipidic carrier system orantigenic liposome).

Thus the present invention is specifically directed towards a targetedlipid vesicles (such as a targeted liposome or micelle), comprising alipid-ligand conjugate of formula I, wherein one or more of groups X₁,X₂, X₃ are a targeting ligand, optionally in combination with otherco-lipids. Alternatively, the present invention is specifically directedtowards an antigenic lipid vesicles (such as an antigenic liposome ormicelle), comprising a lipid-ligand conjugate of formula I, wherein oneor more of groups X₁, X₂, X₃ are an antigenic ligand, optionally incombination with other co-lipids.

In specific embodiments the present invention is also directed towards amixed lipid vesicles (such as a mixed liposome or micelle), comprising alipid-ligand conjugate of formula I, wherein one or more of groups X₁,X₂, X₃ are an antigenic ligand and a targeting ligand, optionally incombination with other co-lipids.

In specific embodiments the lipid vesicles of the invention (i.e.targeted, antigenic or mixed) further comprise one or more bioactiveagents, such as a therapeutic or a diagnostic or an antigenic agent,preferably a therapeutic or a diagnostic agent, either (a) enclosedwithin the internal void of the lipid vesicles of the invention, (b)integrated within the layer(s) or wall(s) of the lipid vesicles of theinvention, for example, by being interspersed among lipids which arecontained within the layer(s) or wall(s) of the lipid vesicles of theinvention, or (c) exposed on the surface of the lipid vesicles of theinvention, whereby the surface exposure is achieved through variouschemical interactions, such as electrostatic interactions, hydrogenbonding, van der Waal's forces or covalent bonding resulting inattachment or adsorption and the like.

A skilled person will understand that all combinations are contemplatedwithin this invention, such as a targeted lipid vesicle comprising anenclosed antigenic agent, etc.

In specific embodiments the lipid vesicles of the invention comprisingan antigenic ligand and/or agent may further comprise one or more,preferably one adjuvant either (a) enclosed within the internal void ofsaid lipid vesicles, (b) integrated within the layer(s) or wall(s) ofsaid lipid vesicles, for example, by being interspersed among lipidswhich are contained within the layer(s) or wall(s) of said lipidvesicles, or (c) exposed on the surface of said lipid vesicles, wherebyits surface exposure is achieved through various chemical interactions,such as electrostatic interactions, hydrogen bonding, van der Waal'sforces or covalent bonding.

Preferably, the one or more adjuvants are enclosed within the internalvoid.

The term “co-lipid” or “vesicle-forming (co-)lipid” as used hereinrefers to lipids which may optionally be present as additional lipids inthe lipid vesicles of the invention and may include acyclic and cyclic,saturated or unsaturated lipids of natural or synthetic origin. As usedherein a co-lipid may be a neutral lipid, a cationic lipid or an anioniclipid. A cationic lipid has a positive net charge and may include lipidssuch as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salts,e.g. the methylsulfate (DOTAP), DDAB, dimethyldioctadecyl ammoniumbromide; 1,2-diacyloxy-3-trimethylammonium propanes, (including but notlimited to: dioleoyl, dimyristoyl, dilauroyl, dipalmitoyl anddistearoyl; also two different acyl chain can be linked to the glycerolbackbone); N-[1-(2,3-dioloyloxy)propyl]-N,N-dimethyl amine (DODAP);1,2-diacyloxy-3-dimethylammonium propanes, (including but not limitedto: dioleoyl, dimyristoyl, dilauroyl, dipalmitoyl and distearoyl; alsotwo different acyl chain can be linked to the glycerol backbone);N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA);1,2-dialkyloxy-3-dimethylammonium propanes, (including but not limitedto: dioleyl, dimyristyl, dilauryl, dipalmityl and distearyl; also twodifferent alkyl chain can be linked to the glycerol backbone);dioctadecylamidoglycylspermine (DOGS);3β-[N—(N′,N′-dimethylamino-ethane)carbamoyl]cholesterol (DC-Chol);2,3-dioleoyloxy-N-(2-(sperminecarboxamido)-ethyl)-N,N-dimethyl-1-propanam-iniumtrifluoro-acetate (DOSPA); β-alanyl cholesterol; cetyl trimethylammonium bromide (CTAB); diC14-amidine;N-tert-butyl-N′-tetradecyl-3-tetradecylamino-propionamidine; 14Dea2;N-(alpha-trimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG);O,O′-ditetradecanoyl-N-(trimethylammonio-acetyl)diethanolamine chloride;1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER);N,N,N′,N′-tetramethyl-N,N′-bis(2-hydroxylethyl)-2,3-dioleoyloxy-1,4-butan-ediammoniumiodide;1-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imidazoliniumchloride derivatives (as described by Solodin et al. (1995) Biochem.43:13537-13544), such as1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM),1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazoliniumchloride (DPTIM), 2,3-dialkyloxypropyl quaternary ammonium compoundderivatives, containing a hydroxyalkyl moiety on the quaternary amine(see e.g. by Feigner et al. J. Biol. Chem. 1994, 269, 2550-2561), suchas: 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI),1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE),1,2-dioleyloxypropyl-3-dimethyl-hydroxypropyl ammonium bromide(DORIE-HP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxybutyl ammoniumbromide (DORIE-HB), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypentylammonium bromide (DORIE-Hpe),1,2-dimyristyloxypropyl-3-dimethyl-hydroxylethyl ammonium bromide(DMRIE), 1,2-dipalmityloxypropyl-3-dimethyl-hydroxyethyl ammoniumbromide (DPRIE), 1,2-disteryloxypropyl-3-dimethyl-hydroxyethyl ammoniumbromide (DSRIE); cationic esters of acyl carnitines (as reported bySantaniello et al. U.S. Pat. No. 5,498,633); cationic triesters ofphospahtidylcholine, i.e. 1,2-diacyl-sn-glycerol-3-ethylphosphocholines,where the hydrocarbon chains can be saturated or unsaturated andbranched or non-branched with a chain length from C₁₂ to C₂₄, the twoacyl chains being not necessarily identical. Neutral or anionic lipidshave a neutral or anionic net charge, respectively. These can beselected from sterols or lipids such as cholesterol, phospholipids,lysolipids, lysophospholipids, sphingolipids or pegylated lipids with aneutral or negative net change. Useful neutral and anionic lipidsthereby include: phosphatidylserine, phosphatidylglycerol,phosphatidylinositol (not limited to a specific sugar), fatty acids,sterols, containing a carboxylic acid group for example, cholesterol,cholesterol sulfate and cholesterol hemisuccinate,1,2-diacyl-sn-glycero-3-phosphoethanolamine, including, but not limitedto, DOPE, 1,2-diacyl-glycero-3-phosphocholines and sphingomyelin. Thefatty acids linked to the glycerol backbone are not limited to aspecific length or number of double bonds. Phospholipids may also havetwo different fatty acids.

A skilled person will understand that the ratio of lipid-ligandconjugates to co-lipids depends on the nature of the bioactive ligand,the nature of the optional bioactive agent enclosed or embedded withinor adsorbed onto or attached to the lipid vesicles, the intended use(treatment of disease, diagnostic assay, etc.), the formulation aspharmaceutical composition and the route of administration.

In one embodiment a lipid vesicle of the invention may compriselipid-ligand conjugates of the invention and other vesicle-forminglipids (co-lipids) preferably in a ratio from 1:1′000 to 1:1, preferably1:500 to 1:50. In further embodiments of the invention, the lipidvesicles may comprises one or more lipid-ligand conjugates wherein theether-lipid of the conjugate comprises unsaturated hydrocarbon chains,which may be crosslinked or polymerized to form polymerized lipidvesicles.

As used herein, the term “polymerized lipid vesicles” and (in particulara polymerized liposome) means a lipid vesicle in which the constituentlipids are covalently bonded to each other by intermolecularinteractions. The lipids can be bound together within a single layer ofthe lipid bilayer (the leaflets) and/or bound together between the twolayers of the bilayer. Polymerizing the lipid layer structure makes theassembly dramatically more resistant to enzymatic breakdown by acids,bile salts or enzymes present in vivo. In addition, controlling thedegree of polymerization and the degradation rate (by choosing specificratios of lipid-ligand conjugates having cleavable or polymerizablehydrocarbon chains), the stability as well as “leakiness” (by generatingpores of a desired size) can be tuned according to the desired escaperate of an optionally enclosed bioactive agent. Thus the design of alipid vesicle allows modulating the optimal escape rate of e.g. anyencapsulated antigen agent at specific immune uptake sites, or anyencapsulated therapeutic agent at specific tissue or cell sites, etc.

As those skilled in the art will recognize, lipidic carrier systems inform of vesicles such as liposomes, micelles, or other vesicles, may bereadily prepared from lipid-ligand conjugates of the invention usingstandard conditions known in the art.

Depending on the desired physical properties, lipid vesicles may beprepared from lipid-ligand conjugates optionally in combination with oneor more co-lipids including stabilizing lipids. The particularstabilizing compounds which are ultimately combined with the presentlipid-ligand conjugates may be selected as desired to optimize theproperties of the resulting lipid vesicles (and are readily identifiableby one skilled in the art without undue experimentation).

Micellar compositions according to the invention may be prepared usingany one of a variety of conventional micellar preparatory methods whichwill be apparent to those skilled in the art. These methods typicallyinvolve suspension of a lipid-ligand conjugate in an organic solvent,evaporation of the solvent, resuspension in an aqueous medium,sonication and centrifugation. The foregoing methods, as well as others,are discussed, for example, in Canfield et al., Methods in Enzymology,Vol. 189, pp. 418-422 (1990); El-Gorab et al, Biochem. Biophys. Acta,Vol. 306, pp. 58-66 (1973); Colloidal Surfactant, Shinoda, et al,Academic Press, N.Y. (1963) (especially “The Formation of Micelles”,Shinoda, Chapter 1, pp. 1-88); Catalysis in Micellar and MacromolecularSystems, Fendler and Fendler, Academic Press, N.Y. (1975). Thedisclosures of each of the foregoing publications are incorporated byreference herein, in their entirety. Optional stabilizing materials becombined with the lipid-ligand conjugates to stabilize the micellarcompositions produced therefrom include lauryltrimethylammonium bromide,cetyltrimethylammonium bromide, myristyltrimethylammonium bromide,(C12-C16)alkyldimethylbenzylammonium chloride, cetylpyridinium bromideand chloride, lauryl sulphate, and the like. Other materials forstabilizing the micellar compositions, in addition to those exemplifiedabove, would be apparent to one skilled in the art based on the presentdisclosure. Liposomal compositions of the invention are particularlypreferred as they are particularly effective as carriers for thedelivery of bioactive agents to tissues and cells or as antigenpresenting carriers.

Liposomal compositions may comprise one or more lipid-ligand conjugatesoptionally in combination with one or more further co-lipids and/or oneor more stabilizing compounds. The lipid-ligand conjugates (andco-lipids) may be in the form of a monolayer or bilayer, and the mono-or bilayer lipids may be used to form one or more mono- or bilayers. Inthe case of more than one mono- or bilayer, the mono- or bilayers aregenerally concentric. Thus, the lipid-ligand conjugates (and co-lipids)may be used to form a unilamellar liposome (comprised of one monolayeror bilayer), an oligolamellar liposome (comprised of two or threemonolayers or bilayers) or a multilamellar liposome (comprised of morethan three monolayers or bilayers).

The selection of suitable co-lipids and stabilizing compounds in thepreparation of liposomal lipid compositions of the invention would beapparent to a person skilled in the art and can be achieved withoutundue experimentation, based on the present disclosure.

Other materials for use in the preparation of liposomal lipidcompositions of the invention, in addition to those exemplified above,would be apparent to one skilled in the art based on the presentdisclosure.

The amount of stabilizing material, such as, for example, additionalamphipathic compound, which is combined with the present lipid-ligandconjugates may vary depending upon a variety of factors, including thespecific lipid-ligand conjugate(s) of the invention selected, thespecific stabilizing material(s) selected, the particular use for whichit is being employed, the mode of delivery, and the like. The amount ofstabilizing material to be combined with the present lipid-ligandconjugates and the ratio of stabilizing material to lipid-ligandconjugates, will vary and is readily determinable by one skilled in theart based on the present disclosure. Typically ratios higher than about4:1, 3:1 or 2:1, of lipid-ligand conjugate to stabilizing lipid, arepreferred.

A wide variety of methods are available in connection with thepreparation of liposomal compositions of the invention. Accordingly, theliposomes may be prepared using any one of a variety of conventionalliposome preparatory techniques which will be apparent to those skilledin the art. These techniques include ethanol injection, thin filmtechnique, homogenizing, solvent dialysis, forced hydration, reversephase evaporation, microemulsification and simple freeze-thawing, Usinge.g. conventional microemulsification equipment. Additional methods forthe preparation of liposomal compositions of the invention from thelipid-ligand conjugates of the present invention include, for example,sonication, chelate dialysis, homogenization, solvent infusion,spontaneous formation, solvent vaporization, controlled detergentdialysis, and others, each involving the preparation of liposomes invarious ways. Typically, methods which involve ethanol injection, thinfilm technique, homogenizing and extrusion are preferred in connectionwith the preparation of liposomal compositions of the invention from thelipid-ligand conjugates of the present invention.

The size of the liposomes can be adjusted, if desired, by a variety oftechniques, including extrusion, filtration, sonication andhomogenization. Other methods for adjusting the size of the liposomesand for modulating the resultant liposomal biodistribution and clearanceof the liposomes would be apparent to one skilled in the art based onthe present disclosure. Preferably, the size of the liposomes isadjusted by extrusion under pressure through pores of a defined size.The liposomal compositions of the invention may be of any size,preferably less than about 200 nanometer (nm) in outside diameter.

As those skilled in the art will recognize, any of the lipid-ligandconjugates and lipidic carrier systems comprising the lipid-ligandconjugates of the invention may be lyophilized for storage, andreconstituted in, for example, an aqueous medium (such as sterile wateror phosphate buffered solution, or aqueous saline solution), preferablyunder vigorous agitation. If necessary, additives may be included toprevent agglutination or fusion of the lipids as a result oflyophilisation. Useful additives include, without limitation, sorbitol,mannitol, sodium chloride, glucose, trehalose, polyvinylpyrrolidone andpoly(ethylene glycol), for example, PEG 400.

C. Nanoparticulate Carrier Systems

Nanoparticulate carrier systems may exist in any shape and anymorphology. Examples of nanoparticulate carrier systems includenanoparticles, nanopowders, nanoclusters, nanocrystals, nanospheres,nanofibers, nanotubes and other geometries. Nanoparticulate vesicularcompositions or nanoparticles are typically small particles havingtypically a diameter of less than 1 micron, preferably in the range ofabout 25-1000 nm, more preferably in the range of about 50-300 nm, mostpreferably in the range of about 60-200 nm. A nanosphere refers to atype of nanoparticle that is approximately spherical in shape and has ahollow core. Typically, nanoparticles have a matrix core structure whichmay be formed using all types of materials and structures, includinginorganic materials, such as metals, and organic materials, such aspolymers including physiologically acceptable polymers. Non-limitingexamples of such polymers include, for example, polyesters (such aspoly(lactic acid), poly(L-lysine), poly(glycolic acid) andpoly(lactic-co-glycolic acid)), poly(lactic acid-co-lysine), poly(lacticacid-graft-lysine), polyanhydrides (such as poly(fatty acid dimer),poly(fumaric acid), poly(sebacic acid), poly(carboxyphenoxy propane),poly(carboxyphenoxy hexane), copolymers of these monomers and the like),poly(anhydride-co-imides), poly(amides), poly(orthoesters),poly(iminocarbonates), poly(urethanes), poly(organophasphazenes),poly(phosphates), poly(ethylene vinyl acetate) and other acylsubstituted cellulose acetates and derivatives thereof,poly(caprolactone), poly(carbonates), poly(amino acids),poly(acrylates), polyacetals, poly(cyanoacrylates), poly(styrenes),poly(vinyl chloride), polyvinyl fluoride), polyvinyl imidazole),chlorosulfonated polyolefins, polyethylene oxide, copolymers,polystyrene, and blends or co-polymers thereof. The nanoparticles mayalso include hydroxypropyl cellulose (HPC), N-isopropylacrylamide(NIPA), polyethylene glycol, polyvinyl alcohol (PVA), polyethylenimine,chitosan, chitin, dextran sulfate, heparin, chondroitin sulfate,gelatin, etc. as well as their derivatives, co-polymers, and mixturesthereof. A non-limiting method for making nanoparticles is describede.g. in U.S. Publication 2003/0138490. In another embodiment the corematerial may be selected from metals, alloys, metalloids, metalcompounds such as metal oxides, inorganic compounds, and carbon-basedmaterials, in particular carbon nanotubes, one-dimensional nanoparticlesof fullerene C₆o, and three-dimensional nanoparticles of fullerene C₇₀.Suitable examples of metals include, but are not limited to, noble or aplatinum metal such as Ag, Au, Pd, Pt, Rh, Ir, Ru, and Os, transitionmetals such as Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, Zr, Nb, Mo, Ta, W, Re,and main group metals such as Al, Ga, In, Si, Ge, Sn, Sb, Bi, Te. Itwill be appreciated that some main group metals, in particular Si andGe, are also commonly referred to as metalloids. Suitable examples ofalloys include, but are not limited to, alloys of noble or platinummetal and transition metals, in particular alloys of silver andtransition metals such as Ag/Ni, Ag/Cu, Ag/Co, and platinum andtransition metals such as Pt/Cu, or noble or platinum alloys such asRu/Pt. Non-limiting examples of inorganic compounds include, but are notlimited, to SiO₂, metal compounds, in particular metal oxides such asTiO₂ and iron oxides. Nanoparticles may also comprise intrinsicfluorescent or luminescent moieties, plasmon resonant moieties, andmagnetic moieties, which provide such nanoparticles with detectableelectrical, magnetic, and/or optical properties.

A skilled person will know that the choice of material may depend on theintended use of the nanoparticle.

In one embodiment, the invention is directed towards a nanoparticlecomprising one or more lipid-ligand conjugates. The one or more lipidconjugates may be entangled, embedded, incorporated, encapsulated,adsorbed or bound to the surface, or otherwise associated with thenanoparticle.

In one specific embodiment the lipid-ligand conjugate may be associatedto a nanoparticle in form of a coating, through intermolecular forcessuch as Van-der-Waals forces, ionic interactions, hydrophobicinteractions, optionally in combination with other co-lipids.

Alternatively, nanoparticles may optionally include one or morefunctional groups, such as, for example, a carboxyl, sulhydryl,hydroxyl, or amino group, for covalently linking one or morelipid-ligand conjugates (or other compounds, such as spacers) to thesurface of the nanoparticles, optionally in combination with otherco-lipids.

Nanoparticles of the invention may also be grouped together (optionallywith a dispersing agent) to form a nanocluster. The independentformulation of each nanoparticle type before cluster formation and aspecial arrangement of nanoparticles within the cluster may allowcontrolling the retention and concentration of a lipid-ligand conjugateand thus of the bioactive agent.

In some embodiments, the nanoparticles may further comprise anadditional bioactive agent entrapped, embedded, or encapsulated withinthe solid matrix core of the nanoparticle.

In preferred embodiments, the lipid-ligand coated nanoparticles may beformed from nanosized core particles and one or more lipid-ligandconjugates of the present invention and optionally one or moreco-lipids. In any given lipid-ligand coated nanoparticle, thelipid-ligand conjugates may be in the form of a monolayer or a bilayer.In the case of more than one mono- or bilayer, the mono- or bilayers aregenerally concentric. Coating of the nanoparticles is preferably carriedout in a solution comprising the lipid-ligand conjugates of theinvention and by allowing sufficient time to allow the lipid-ligandconjugates to coat the nanoparticles.

In some embodiments, the one or more ligands of the one or morelipid-ligand conjugates are one or more antigenic ligands.

The amount of antigenic ligand per nanoparticle (or surface density ofthe antigenic ligand) to induce an immune response depends on manyfactors, such as the nature of the immune response itself (humoral vs.cell-mediated), the immunogenicity of the antigen ligand, theimmunogenic constitution of the challenged organism, and theadministration route and duration of exposure to the antigen. Clearly,immunization of a subject may be enhanced by the use of multiple copiesof an antigen as a multivalent display thereby increasingsite-specifically antigen concentration and thus inducing a long-lastingimmune responses. It is particularly desirable in case of antigenligands such as small peptides or carbohydrates, that are difficult toadminister and generally fail to elicit an effective immune response dueto the hapten-related size issues.

Thus, in some embodiments the nanoparticle displays single or multiplecopies of one antigen ligand or a combination of different antigenligands on its surface (in form of a multivalent display). As usedherein, the term “multivalent” refers to the display of more than onecopy or type of antigen on a carrier system.

More specifically, the present invention relates to a nanoparticlecomprising a solid core which is coated by at least one lipid-ligandconjugate of formula I, wherein one or more of X₁, X₂, X₃ are anantigenic ligand, and optionally other matrix or co-lipids.

Immunization may be further improved by including targeting ligands todirect the nanoparticle to the appropriate immune cell or location.Compounds which may act as targeting ligands are compounds thatinterfere with the adherence of pathogens to host cells and thussuccessful colonization. Examples of such compounds may include thetetanus toxoid; P pili of E. coli; type IV pili of Pseudomonasaeruginosa, Neisseria species, Moraxella species, EPEC, or Vibriocholerae; fimbrial genes and several a fimbrial adhesins, including FHA,pertactin, pertussis toxinand BrkA of Bordetella pertussis; and SipB-Dof Salmonella typhimurium; and the adenovirus adhesion; the Reovirussigma-1 protein which targets the M-cell.

Thus, the invention also refers to a nanoparticle comprising a solidcore which is coated by at least one lipid-ligand conjugate of formulaI, wherein one or more of X₁, X₂, X₃ are an antigenic ligand and/or atargeting ligand, and optionally other matrix or co-lipids.

In other embodiments a lipid-ligand coated nanoparticle furthercomprises a single antigenic agent or a combination of antigenic agents(multivalent) enclosed or embedded within the solid core of thenanoparticle. Thus, the invention also refers to a nanoparticlecomprising a solid core, which is coated by at least one lipid-ligandconjugate of formula I, wherein one or more of X₁, X₂, X₃ are anantigenic ligand and/or a targeting ligand, and optionally other matrixor co-lipids, and wherein the solid core optionally comprises one ormore further antigenic agents.

In yet other embodiments the nanoparticle further comprises one or moreadjuvants enclosed, embedded or dispersed within the solid core of thenanoparticle.

As used herein the term “adjuvant” refers to any material capable ofenhancing a humoral and/or cellular immune response to a specificantigen. Suitable adjuvants may be displayed on the surface of ananoparticle, intercalated into a nanoparticle wall or encapsulated intoa nanoparticle interior. Examples of adjuvants that may be used topromote the production of serum and/or mucosal antibodies as well ascell-mediated immune responses against co-administered antigens includeE. coli heat-labile enterotoxin holotoxin (LT) and Vibrio choleraeenterotoxin (CT) as well as the KPL adjuvant (derived from the cell wallof Salmonella Minnesota).

As used herein, the terms “displayed” or “surface exposed” refer to anyligand that is present at the external surface of a carrier system suchas a lipidic vesicle or a nanoparticle and thus is accessible forrecognition. A variety of diseases and disorders may be treated by suchnanoparticle vaccine constructs or assemblies, including: inflammatorydiseases, infectious diseases, cancer, genetic disorders, organtransplant rejection, autoimmune diseases and immunological disorders.

Thus the invention also encompasses a vaccine comprising multivalentnanoparticles comprising a solid core and one or more surface exposedlipid-ligand conjugates, wherein the ligand is one or more antigenicand/or targeting ligands, further optionally comprising an adjuvantand/or a further antigenic agent embedded in the solid core of thenanoparticles. In further embodiments, the one or more ligands of theone or more lipid-ligand conjugates are one or more therapeutic ordiagnostic agent. Methods of production of a nanoparticle of theinvention comprising a surface exposed lipid-ligand conjugate includethe steps of (a) providing a nanoparticle and (b) associating the one ormore lipid-ligand conjugates to the nanoparticle through adsorption orattachment to form a lipid-ligand coated nanoparticle. Alternatively themethods include the steps of (a) providing a nanoparticle, (b)associating the one or more ether-lipids to the nanoparticle throughadsorption or attachement to form a lipid-coated nanoparticle and (c)covalently linking the one or more bioactive ligands to the one or moreether-lipids associated with the surface of the nanoparticle to form alipid-ligand coated nanoparticle. The (preformed) lipid-coatednanoparticles are part of an application filed concurrently, which isincorporated herein in its entirety.

Typical methods to fabricate nanoparticles of suitable size includevaporization methods (e.g., free jet expansion, laser vaporization,spark erosion, electro explosion and chemical vapor deposition),physical methods involving mechanical attrition (e.g., the pearlmillingtechnology, Elan Nanosystems, Ireland), and interfacial depositionfollowing solvent displacement.

In further embodiments, the invention is also directed towards ananosphere comprising one or more lipid-ligand conjugates. As opposed toa nanoparticle, a nanosphere as a hollow interior, which may easily beused to enclose and subsequently deliver one or more bioactive agents tocells or tissues of interest. The release rate of such encapsulatedbioactive agent(s) can be modulated, for example, by known techniques.

D. Pharmaceutical Compositions and Formulations

The carrier systems of the invention may be present as a pharmaceuticalcomposition, e.g. which further comprises a pharmaceutically acceptablediluents, excipient or carrier, such as physiological saline orphosphate buffer, selected in accordance with the route ofadministration and standard pharmaceutical practice.

Thus in a further aspect the present invention is directed towards apharmaceutical composition comprising one or more lipidic ornanoparticulate carrier system comprising ligand-lipid conjugatesoptionally in combination with other co-lipids and pharmaceuticallyacceptable diluents, excipient or carrier.

Preferably the lipidic carrier system is lipid vesicle, such as aliposome or a micelle and the nanoparticulate carrier system is ananoparticle or nanosphere.

It is understood that the term “one or more lipid-ligand conjugates”refers to all possible embodiments as disclosed herein, i.e. conjugateswherein the ligand is one or more of a targeted, antigenic, therapeuticand diagnostic ligand and mixtures thereof. Optionally a further one ormore bioactive agent is enclosed or embedded within or adsorbed onto orattached to the lipidic or nanoparticulate carrier system.

The pharmaceutical compositions of the present invention can be used ineither in vitro, such as cell culture applications, or in vivoapplications. With respect to in vivo applications, the lipidformulations of the present invention can be administered to a patientin a variety of forms adapted to the chosen route of administration,including parenteral, oral, or intraperitoneal administration.Parenteral administration includes intravenous, intramuscular,interstitially, intraarterially, subcutaneous, intraocular,intrasynovial, transepithelial (including transdermal), pulmonary viainhalation, ophthalmic, sublingual and buccal, topically (includingophthalmic, dermal, ocular, rectal), and nasal inhalation viainsufflation administration, preferably intravenous administration.

The useful dosage to be administered and the particular mode ofadministration will vary depending upon the therapeutic or diagnosticuse contemplated, the particular bioactive agent and lipid compound usedas well as the form of the carrier system, e.g. micelle, liposome ornanoparticle, as well as factors such as age, weight, physical conditionof the subject to be treated, as will be readily apparent to thoseskilled in the art. The use of targeted pharmaceutical compositionsaccording to the invention allows administration of lower dosages forthe desirable therapeutic effect to be achieved.

By way of general guidance, the ratio of lipid-ligand conjugate in thecarrier system will vary from between 0.05 to 5 mole %, with a ratio of0.1 to 2 mole % being more preferred, and between about 0.01 mg andabout 10 mg of the particular antigenic, therapeutic or diagnostic agenteach per kilogram of patient body weight, may be suitable to beadministered, although higher and lower amounts can be used.

E. Methods of Use

As indicated above, in one specific embodiment the targeted lipid-ligandconjugates and in particular the targeted vesicles (i.e. liposomes andmicelles) and targeted nanoparticles comprising these, as well as therespective pharmaceutical compositions thereof, are particularlysuitable for use as carriers for a targeted delivery of one or morebioactive agents, preferably therapeutic, diagnostic and/or antigenicagents.

Thus, the targeted lipid-ligand conjugates of the present invention areparticularly applicable for use in vitro and/or in vivo in methods forthe treatment of diseases, for which a targeted delivery of one or morespecific bioactive agents, preferably therapeutic, diagnostic and/orantigenic agents, to tissues or cells is desirable or required. In thecase of targeted nanoparticles and pharmaceutical compositions thereof,the one or more bioactive agent is preferably entrapped within the solidcore.

In the case of targeted lipid vesicles and pharmaceutical compositionsthereof, the one or more bioactive agent is preferably enclosed withinthe internal void, or incorporated into the lipid bilayer.

In further aspects, the present invention also encompasses methods fortransport of a diagnostic or biologically active compound across amembrane, in particular methods for intracellular delivery of one ormore bioactive agent which comprises contacting cells with apharmaceutical composition of the invention.

In another specific embodiment the antigenic vesicles (i.e. liposomesand micelles) and antigenic nanoparticles comprising these, as well asthe respective pharmaceutical compositions thereof, are particularlysuitable for use as antigen display systems. Thus, the antigeniclipid-ligand conjugates of the present invention are particularlyapplicable e.g. for use in immunization methods and/or for in vitro/invivo diagnostic applications. Optionally the antigenic vesicles (i.e.liposomes and micelles) and antigenic nanoparticles may further compriseone or more bioactive agents. In case of antigenic nanoparticles, theone or more bioactive agent is preferably entrapped within the solidcore. In the case of targeted lipid vesicles and pharmaceuticalcompositions thereof, the one or more bioactive agent is preferablyenclosed within the internal void, or incorporated into the lipidbilayer.

Thus, in yet another aspect the present invention is directed towards anantigen display system for prophylactic and therapeutic vaccines whichcomprises an antigenic lipid vesicle or an antigenic nanoparticlecomprising one or more antigenic lipid-ligand conjugates optionally incombination with other co-lipids, wherein the optionally comprises oneor more adjuvants and/or one or more bioactive agents.

Also encompassed by the present invention are methods for triggering ormodulating an immune response to an antigen in a subject which comprisesthe display of antigens to antigen presenting cells, in particular todendritic cells, macrophages, B-cells and endothelial cells, andadministering subsequently said antigen presenting cells to the subject.Other aspects of the invention include methods for transport of abiologically active compound across a membrane and/or methods ofdelivery of a biologically active compound into a cell using carriersystems of the invention.

Further applications that are contemplated include e.g. in vitroapplication for growth promotion and differentiation of cells as well asmodification of expression profiles and post-translational modificationpatterns of biological products manufactured in bioreactors.

F. Kits

In yet a further aspect, the present invention relates to a kitcomprised of a container that is compartmentalized for holding thevarious elements of the kit. One compartment may contain a predeterminedquantity of either lipid-ligand conjugate or a carrier systems preparedtherefrom In case of carrier systems such as liposomes, these may bewith or without a pH buffer to adjust the composition pH tophysiological range of about 7 to about 8, or else in lyophilized orfreeze dried form for reconstitution at the time of use. Also includedwithin the kit will be other reagents and instructions for use.

The present invention is further described in the following examples.

EXAMPLES

Materials:

1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) is from Merck & Cie(Schaffhausen, Switzerland). Cholesterol, DOPE, DSPC, POPC,MPEG2000-DOPE (880130), and fluorescent lipids NBD-DOPE(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)(ammonium salt)) (810145P) and PhB-DOPE (810150) are purchased fromAvanti Polar Lipids (Alabaster, Ala.). Functionalized PEG propionic acid(PA) derivative Fmoc-NH-PEG₁₂-PA-COOH (851024) is obtained fromNovabiochem, Fmoc-NH-PEG₈-PA-COOH (PEG1830), Fmoc-NH-PEG₃₆-PA-COOH(PEG4400) and MPEG(2 kDa)-amine (PEG1152) from IRIS Biotech GmbH.Diphenyldiazomethane resin (D-2230) is obtained from Bachem AG,H-Thr(tBu)-2-CITrt resin (RRA-1251) from CBL Patras, H-Gly-2-CITrt resin(856053) and Sieber resin (855008) from Novabiochem. All other chemicalsand solvents are A.R. grade or above.

Aza-peptide Michael acceptor trans-Cbz-D-Ala-D-Ala-2-aza-Asn-acrylicacid (RR11a-OH) and its activated ester with N-hydroxysuccinimide(RR11a-NHS) are synthesized by WuXi AppTec Co. Ltd. (Ekici et al, 2004,J. Med. Chem. 47, 1889-1892; Reisfeld et al., Nanomedicine:Nanotechnology, Biology and Medicine 7, Issue 6, 2011, 665-673).2,3-Bis(tetradecyloxy)propan-1-amine is synthesized according to Kokotoset al. Chemistry-A European Journal, 2000, vol. 6, #22, 4211-4217. In ananalogous way bis(3-((Z)-octadec-9-enyloxy)propyl)amine is obtained fromoleyl methanesulfonate and bis(3-hydroxypropyl)amine (see MaGee et al.,J. Journal of Organic Chemistry, 2000, vol. 65, #24, 8367-8371).

Cell Culture:

M21 human melanoma cells, obtained from Cell Culture Strain Collection,Merck KGaA, Darmstadt, Germany are maintained at 37° C. with 5% CO₂ inDMEM with High Glucose culture medium (Life Technologies, Carlsbad,Calif.) supplemented with 10% fetal bovine serum. Cells are regularlypassaged and plated in 6-well culture plates for 16 hours beforeexperiment at 0.3×10⁶ cells in 2 ml medium. The M21 cells are incubatedwith liposomes in Opti-MEM serum free culture medium for 1 hour at 37°C., and then harvested using Cell Dissociation Buffer (LifeTechnologies, Carlsbad, Calif.) after one time wash with Opti-MEM.Colocalization of NBD-DOPE is determined by Guava easyCyte 8HT (EMDMillipore Corp., Billerica, Mass.).

Statistical Analysis:

Statistical analyses are preformed with Student's t-test. Differencesamong means are considered to be statistically significant at a p valueof <0.01.

Example 1: Synthesis of(2S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-glutamicacid-α-tert-butylester-γ-2,3-bis(tetradecyloxy)propyl-amide

15 g of Fmoc-Glu(OSu)OtBu((2S)—N_(□)-(9-fluorenylmethyloxycarbonyl)-glutamic acidα-tert-butyl-ester γ-N-hydroxysuccinimide ester) are dissolved indichloromethane at room temperature. After addition of 15.3 g of2,3-bis(tetradecyloxy)propan-1-amine, the mixture is stirred for 17hours and evaporated to dryness. The residue is dissolved in a minimumamount of dichloromethane and purified by column chromatography usingSiO₂ as solid phase and methyl tert. butylether/hexane/7:3 as eluent.After evaporation of product fractions 25.5 g of(2S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-glutamicacid-α-tert-butylester-γ-2,3-bis(tetradecyloxy)propyl-amide are obtainedas a colorless solid. ¹H-NMR in CDCl₃ (TMS as internal standard),chemical shift in ppm: 7.76 (d, 2H, Fmoc), 7.61 (d, 2H, Fmoc), 7.25-7.43(m, 4H, Fmoc), 6.13 (bs, NH, 1H), 5.60 (bs, NH, 1H), 4.39, 4.18-4.25 (dand m, 4H), 3.21-3.62 (m, 9H), 1.97-2.23 (m, 4H), 1.51-1.60 (m, 4H),1.47 (s, 9H), 1.25 (m, 44H, CH₂), 0.84-0.91 (m, 6H, 2× alkyl-CH₃).

Example 2: Synthesis of(2S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-glutamicacid-γ-2,3-bis(tetradecyloxy)propyl-amide

In a 100 ml flask 4.6 g (5.1 mmol)(2S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-glutamicacid-α-tert-butylester-γ-2,3-bis(tetradecyloxy)propyl-amide aredissolved in 25 ml dichloromethane and treated with 25 mltrifluoroacetic acid. After 1 h the ester group is completely cleavedand the solution is poured onto 50 ml of cold water.

The organic layer is extracted, washed to neutral pH with water anddried over Na₂SO₄. The organic layer is filtered off and the solventevaporated to afford 4.2 g of the desired product (5.0 mmol, 98% yield,TLC: MtBE/hexane 7:3; Rf=0.43.

Example 3: Synthesis of (2S)-glutamicacid-γ-(2,3-bis(tetradecyloxy)propyl)amide

5 g of (2S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-glutamicacid-α-tert-butylester-γ-2,3-bis(tetradecyloxy)propyl-amide are added to85 ml of N,N-dimethylformamide. To the mixture 2.6 ml of piperidine areadded. The mixture is stirred for three hours at room temperature andthen evaporated to dryness under vacuum to give 5.2 g of (2S)-glutamicacid-γ-(2,3-bis(tetradecyloxy)propyl)amide as a colorless solid, whichcan be used in the preparation of lipidic vesicles or for priorderivatization with an active agent or a spacer group.

Example 4: Synthesis of(R)-2-amino-N1-(2-(4-methoxybenzamido)ethyl)-N4,N4-bis(3-((Z)-octadec-9-enyloxy)propyl)succinamide(a) Synthesis of N-(2-aminoethyl)-4-methoxybenzamide

3.0 g 4-Methoxybenzoyl chloride are added to 30 mL 1,2-diaminoethane indichloromethane at −78° C. and subsequently allowed to warm to 23° C. Anaqueous acid-base workup and evaporation to dryness under vacuum give1.65 g of N-(2-aminoethyl)-4-methoxybenzamide, a pale yellow oil. ¹H-NMRin CDCl₃ (TMS as internal standard), chemical shift in ppm: 8.53 (t, 1H,NH), 7.91 (d, 2H, Benz), 6.99 (d, 2H, Benz), 4.75 (bs, 2H, NH₂), 3.81(s, 3H, CH₃), 3.39, (dd, 2H, CH₂), 2.82 (t, 2H, CH₂).

(b) Synthesis of (R)-tert-butyl3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(2-(4-methoxybenzamido)ethylamino)-4-oxobutanoate

3.0 g 2 N-(2-aminoethyl)-4-methoxybenzamide (obtained in step (a)) and1.70 mL N-methylmorpholine in DMF (0° C.) are added to a solution of6.35 g Fmoc-Asp(OtBu)-OH, 1.70 mL N-methylmorpholine and 2.00 mLisobutylchloroformate in ethylacetate (−12° C.) and stirred for 3 hwhile allowing to warm to 23° C. Dilution of the resulting suspensionwith ethylacetate, followed by an aqueous acid-base workup andevaporation to dryness under vacuum yield 9.55 g (R)-tert-butyl3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(2-(4-methoxybenzamido)ethylamino)-4-oxobutanoate.This crude material is suspended in isopropylether for 23 h, thenfiltered off and dried to furnish 4.47 g (R)-tert-butyl3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(2-(4-methoxybenzamido)ethylamino)-4-oxobutanoateas white crystals. ¹H-NMR in CDCl₃ (TMS as internal standard), chemicalshift in ppm: 8.28 (t, 1H, NH), 8.07 (t, 1H, NH), 7.89 (d, 2H, Fmoc),7.81 (d, 2H, Benz), 7.71-7.60 (m, 2H, Fmoc and 1H, NH), 7.46-7.27 (m,4H, Fmoc), 6.96 (d, 2H, Benz), 4.35-4.20 (m, 3H, Fmoc, and 1H CH), 3.78(s, 3H, CH₃), 3.40-3.20, (m, 4H, 2×CH₂), 2.69 (dd, 1H, CH₂), 2.46 (dd,1H, CH₂), 1.37 (s, 9H, 3×CH₃).

(c) Synthesis of(R)-3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(2-(4-methoxybenzamido)ethylamino)-4-oxobutanoicsodium acetate

To 3.0 g (R)-tert-butyl3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(2-(4-methoxybenzamido)ethylamino)-4-oxobutanoate(obtained in step (b)) in dichloromethane 30.0 mL trifluoroacetic acidare added at 23° C. Upon completion of the reaction aq. NaHCO₃ is addedto furnish a white precipitate which is washed with dichloromethane anddried to yield 2.55 g(R)-3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(2-(4-methoxybenzamido)ethylamino)-4-oxobutanoicsodium acetate as a white powder. ¹H-NMR in SO(CD₃)/CD₃OD, 1:1, (TMS asinternal standard), chemical shift in ppm: 7.85-7.79 (m, 2H, Fmoc and2H, Benz), 7.68 (d, 2H, Fmoc), 7.45-7.29 (m, 4H, Fmoc), 6.93 (d, 2H,Benz), 4.51-4.17 (m, 3H, Fmoc and 1H, CH), 3.78 (s, 3H, CH₃), 3.47-3.34,(m, 4H, 2×CH₂), 2.82 (dd, 1H, CH₂), 2.63 (dd, 1H, CH₂).

(d) Synthesis of (9H-fluoren-9-yl)methyl(R,Z)-1-(4-methoxyphenyl)-10-(3-((4-octadec-9-enyloxy)propyl)-1,6,9-trioxo-14-oxa-2,5,10-triazadotriacont-23-en-7-ylcarbamate

0.48 g(R)-3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(2-(4-methoxybenzamido)ethylamino)-4-oxobutanoicsodium acetate (obtained in step (c)) in dimethylformamide are cooled to10° C. and then 0.46 g bis(3-((Z)-octadec-9-enyloxy)propyl)amine, 0.37 gCOMU and 0.20 g DIPEA are added subsequently. After stirring at 23° C.for 20 h the solution is filtered through a pad of Alox and this rinsedwith little dimethylformamide. The filtrate is diluted withethylacetate, washed with water and evaporation to dryness under vacuumgive 1.12 g orange oil which was purified by column chromatography toyield 0.41 g (9H-fluoren-9-yl)methyl(R,Z)-1-(4-methoxyphenyl)-10-(3-((Z)-octadec-9-enyloxy)propyl)-1,6,9-trioxo-14-oxa-2,5,10-triazadotriacont-23-en-7-yl-carbamate.¹H-NMR in CDCl₃ (TMS as internal standard), chemical shift in ppm: 7.86(d, 2H, Benz), 7.69 (d, 2H, Fmoc), 7.55 (d, 2H, Fmoc), 7.42-7.23 (m, 4H,Fmoc and 1H, NH), 6.88 (d, 2H, Benz and 1H, NH), 6.12 (bd, 1H, NH),5.41-5.26 (m, 4H, 4×CH), 4.60-4.33 (m, 3H, Fmoc), 4.17 (t, 1H, CH), 3.82(s, 3H, CH₃), 3.62-3.23, (m, 16H, 8×CH₂ and 1H, CH₂), 2.73 (dd, 1H,CH₂), 2.05-1.95 (m, 8H, 4×CH₂), 1.85-1.65 (m, 4H, 2×CH₂), 1.57-1.45 (m,4H, 2×CH₂), 1.24 (bs, 44H, 22×CH₂), 0.88 (t, 6H, 2×CH₃).

(e) Synthesis of(R)-2-amino-N1-(2-(4-methoxybenzamido)ethyl)-N4,N4-bis(3-((Z)-octadec-9-enyloxy)propyl)succinamide

To 2.12 g (9H-fluoren-9-yl)methyl(R,Z)-1-(4-methoxyphenyl)-10-(3-((Z)-octadec-9-enyloxy)propyl)-1,6,9-trioxo-14-oxa-2,5,10-triazadotriacont-23-en-7-yl-carbamate(obtained in step (d)) in dichloroethane 0.75 g diethylamine is added,stirred for 26 h followed by evaporation to dryness under vacuum to give1.90 g crude material which is purified by adsorption to 20 g DowexMonosphere and subsequent desorption by ammonia in ethanol to yield 1.09g(R)-2-amino-N1-(2-(4-methoxybenzamido)ethyl)-N4,N4-bis(3-((Z)-octadec-9-enyloxy)propyl)succinamide.¹H-NMR in CDCl₃ (TMS as internal standard), chemical shift in ppm: 7.88(d, 2H, Benz and 1H, NH), 7.64 (t, 1H, NH), 6.89 (d, 2H, Benz),5.42-5.26 (m, 4H, 4×CH), 3.82 (s, 3H, CH₃), 3.65-3.49, (m, 4H, 2×CH₂),3.42-3.28 (m, 12H, 6×CH₂ and 1H, CH), 2.99 (dd, 1H, CH₂), 2.71 (dd, 1H,CH₂), 2.10-1.92 (m, 8H, 4×CH₂ and 2H, NH₂), 1.85-1.67 (m, 4H, 2×CH₂),1.60-1.47 (m, 4H, 2×CH₂), 1.28 (bs, 44H, 22×CH₂), 0.90 (t, 6H, 2×CH₃).MS: 947.9 [M+Na]⁺.

Example 5: Synthesis of N²,N,N-dimethylaminomethylene-10-formyl-folicacid-α-tert. butyl ester-vγ2,3 bis(tetradecyloxy) propylamide

2.2 g of N²,N,N-dimethylaminomethylene-10-formyl-pteroic acid are addedto 46 ml of N,N-dimethylformamide. After addition of 3.2 g of0-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate themixture is stirred for 20 minutes at room temperature. Then a mixture of5.0 g of (2S)-glutamic acid-γ-(2,3-bis (tetradecyloxy)propyl)amide and50 ml N,N-dimethylformamide is added dropwise. After stirring at roomtemperature for 17 hours, the solids are removed by filtration and thefiltrate is evaporated to dryness in vacuum at 40° C. The residue isdissolved in 100 ml of dichloromethane. The dichloromethane solution iswashed with 25 ml of aqueous citric acid solution, 25 ml of aqueous 5%sodium hydrogen carbonate solution and 25 ml of water. Each of theaqueous phases is extracted with dichloromethane. The combineddichloromethane phases are dried over magnesium sulphate, evaporated todryness to give a yellow foam which is dissolved in a mixture ofdichloromethane/methanol/95:5 and stirred for 15 min. at 40° C. Solidsare removed by filtration and the filtrate is purified by columnchromatography using SiO₂ as solid phase anddichloromethane/methanol/95:5 as eluent. After evaporation of productfractions 2.7 g of a yellow foam are obtained which are again purifiedby column chromatography as described above to give 2.2 g ofN²,N,N-dimethylaminomethylene-10-formyl-folic acid-α-tert. butylester-α-(2,3 bis(tetradecyloxy)propyl) amide as a pale yellow foam.¹H-NMR in CDCl₃ (TMS as internal standard), chemical shift in ppm: 10.00(bs, 1H, N3-H), 8.96 (s, 1H, C7-H), 8.76, 8.72 (2s, 2H, CHN, CHO), 7.88(d, 2H, C2′-H, C6′-H), 7.73 (d, 1H, NH(Glu)), 7.35 (d, 2H, C3′-H,C5′-H), 6.26 (d, 1H, C□-NH), 5.32 (s, 2H, C6-H₂), 4.53 (m, 1H, CH-Glu),3.30-3.56 (m, 9H, m, 4CH₂, CH—O-alkyl), 3.22 (s, 3H, N—CH₃), 3.15 (s,3H, N—CH₃), 2.03-2.40 (m, 4H, 2 CH₂-Glu), 1.54 (m, s, 4H, 2CH₂), 1.46(s, 9H, OC(CH₃)₃), 1.24 (s, 44H, 22 CH₂), 0.87 (m, 6H, 2x alkyl-CH₃).

Example 6: Synthesis of folic acid-γ-(2,3 bis(tetradecyloxy)propyl)amide

2.1 g of N²,N,N-dimethylaminomethylene-10-formyl-γ folic acid-α-tert.butyl ester-γ-2,3 bis(tetradecyloxy)propylamide are dissolved in 105 mldichloromethane. After addition of 105 ml of trifluoroacetic acid themixture is stirred for 1 hour at room temperature and then evaporated todryness at 40° C. to give 3.4 g of a yellow foam. The latter isdissolved in 105 ml of tetrahydrofuran and 105 ml of 1M aqueous sodiumhydroxide solution are added dropwise while stirring. The mixture isheated to 50° C. for 2.5 hours. After cooling to room temperature theorganic layer is separated and evaporated to dryness. To the residue 105ml of dichloromethane and 105 ml of 1 M aqueous hydrochloric acid areadded. The mixture is stirred for 5 minutes at room temperature and theprecipitated product is sucked off, washed with 500 ml of water and thendried at 40° C. in vacuum to 1.76 g of folic acid-γ-(2,3bis(tetradecyloxy)propyl)amide as a yellow solid. ¹H-NMR in DMSO-d₆ (TMSas internal standard), chemical shift in ppm: 11.59 (bs, 1H, N3-H), 8.64(s, 1H, C7-H), 8.17 (d, 1H, NH), 7.81 (t, 1H, NH), 7.66 (d, 2H, C2′-H,C6′-H), 7.01 (bs, NH, 1H), 6.92 (t, 2H, NH), 6.64 (d, 2H, C3′-H, C5′-H),4.49 (d, 2H, C6-H₂), 4.29 (m, 1H, CH-Glu), 3.26-3.46 (m, 5H, 2CH₂,CH—O-alkyl), 3.08 (s, 2H, CH₂), 2.17-2.2.5 (m, 2H, CH₂), 1.84-2.11 (2m,2H, CH₂), 1.42, 1.23 (m, s, 44H, 22 CH₂), 0.85 (m, 6H, 2x alkyl-CH₃).

Example 7: Synthesis of(2S,47S)-47-[2-N-(dimethylamino)methylene]-10-formylpteroylamino-2-[3-[[2,3-bis(tetradecyloxy)propyl]amino]-3-oxopropyl]-4,44-dioxo-7,10,13,16,19,22,25,28,31,34,37,40-dodecaoxa-3,43-diazaoctatetracontane-1,48-dioicacid

(a) Synthesis of Fmoc-Glu(DMA)-diphenylmethyl resin

In a 100 ml SPPS reactor 3.85 g of diphenyldiazomethane resin (3.3 mmol)are washed twice with 30 ml DCM and treated with a solution of 4.2 g of2S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-glutamicacid-γ-2,3-bis(tetradecyloxy)propyl-amide (see example 2, 1.5 eq., 5.0mmol) in 30 ml DCM over night. The solution is filtered off and theresin is washed with DCM four times. To destroy eventually un-reacteddiphenyldiazomethane the resin is treated with 125 μl acetic acid (0.5eq., 2.2 mmol) in 30 ml DCM for 15 minutes and washed afterwards threetimes alternating with 30 ml dimethylformamide and isopropanol. Theresin is washed twice with diisopropyl ether and dried over night invacuo. 6.7 g of the desired product are obtained (>100% of theory, yieldin theory 6.5 g). The loading of the resin is determined to 0.49 mmol/gby UV measurement of the Fmoc cleavage product at 304 nm (maximumloading in theory 0.51 mmol/g).

(b) Synthesis of H-Glu-OtBu-NH-PEG₁₂-PA-Glu(DMA)-diphenylmethyl resin

H-Glu-OtBu-NH-PEG₁₂-PA-Glu(DMA)-diphenylmethyl resin is obtained throughconventional solid phase synthesis by the following reaction sequence:

(1) cleavage of the Fmoc group of the Fmoc-Glu(DMA)-diphenylmethyl resinwith piperidin in DMF,

(2) condensation with Fmoc-NH-PEG₁₂-PA-COOH using HBTU in DMF and DIPEA,

(3) cleavage of the Fmoc group of theFmoc-NH-PEG₁₂-PA-Glu(DMA)-diphenylmethyl resin with piperidin in DMF,

(4) condensation with Fmoc-Glu-OtBu using HBTU in DMF and DIPEA andfinally

(5) cleavage of the Fmoc group of theFmoc-Glu-OtBu-NH-PEG₁₂-PA-Glu(DMA)-diphenylmethyl resin with piperidinin DMF.

(c) Synthesis of[2-N-(dimethylamino)methylene]-10-formylpteroyl-Glu-OtBu-NH-PEG₁₂-PA-Glu(DMA)-diphenylmethylresin

[2-N-(dimethylamino)methylene]-10-formylpteroyl-Glu-OtBu-NH-PEG₁₂-PA-Glu(DMA)-diphenylmethylresin is obtained through conventional solid phase synthesis by reactingH-Glu-OtBu-NH-PEG₁₂-PA-Glu(DMA)-diphenylmethyl resin in DMF with[2-N-(dimethylamino)methylene]-10-formylpteroic acid, HATU and DIPEA.

(d) Synthesis of(2S,47S)-47-[2-N-(dimethylamino)methylene]-10-formylpteroylamino-2-[3-[[2,3-bis(tetradecyloxy)propyl]amino]-3-oxopropyl]-4,44-dioxo-7,10,13,16,19,22,25,28,31,34,37,40-dodecaoxa-3,43-diazaoctatetracontane-1,48-dioicacid

4.5 g[2-N-(dimethylamino)methylene]-10-formylpteroyl-Glu-OtBu-NH-PEG₁₂-PA-Glu(DMA)-diphenylmethylresin are washed with 45 ml dichloromethane, filtered off and suspendedagain in 45 ml dichloromethane. Then 41.4 ml of trifluaroacetic acid areadded followed by 2.25 ml triisopropylsilane. The suspension is stirredat room temperature for 1 hour and then filtered. The resin is washedthree times with 45 ml dichloromethane each. The combined filtrates areevaporated in vacuo to yield 5.75 g of an amber oil. HPLC: 90.7% area,ESI-MS: monoisotopic M_(W calc).=1718.1, M_(W) [M−H]⁻=1718.0.

Example 8: Synthesis of(2S,47S)-47-pteroylamino-2-[3-[[2,3-bis(tetradecyloxy)propyl]amino]-3-oxopropyl]-4,44-dioxo-7,10,13,16,19,22,25,28,31,34,37,40-dodecaoxa-3,43-diazaoctatetracontane-1,48-dioicacid

4.6 g(2S,47S)-47-[2-N-(dimethylamino)methylene]-10-formylpteroylamino-2-[3-[[2,3-bis(tetradecyloxy)propyl]amino]-3-oxopropyl]-4,44-dioxo-7,10,13,16,19,22,25,28,31,34,37,40-dodecaoxa-3,43-diazaoctatetracontane-1,48-dioicacid are stirred with 460 ml 1N NaOH at 50° C. for 2 hours. The reactionmixture is brought to pH 12.5 by the addition of 59.2 g 32% ic NaOH. Thebrown solution is treated with 0.46 g activated carbon for 15 min at 50°C., filtered hot and brought to pH 1 by the addition of 3.2 g 37% icHCl. The resulting precipitate is collected by filtration, washed withwater and dried at room temperature in vacuo to yield 1.2 g of agreenyellow solid. H PLC: 89.9% area, ESI-MS: monoisotopicM_(W calc).=1635.0, M_(W) [M−H]⁻=1634.1.

Example 9: Synthesis of RGD Lipid PentapeptideCyclo[-Asp-hGlu(DMA)-D-Val-Arg-Gly-]

(a) Synthesis of Fmoc-hGlu(OBzI)-OH

Commercially available homo glutamic acid (H-hGlu-OH) is side chainprotected as δ-benzyl ester following a published protocol (Benoiton L.,Can. J. Chem., 40, 570 (1962)) (yield: 19.8 g, 26% of theory, TLC(CHCl₃/MeOH/32% acetic acid 5:3:1) R_(f)=0.62). Without furtherpurification H-hGlu(OBzI)-OH (19.7 g, 77.6 mmole) is dissolved in amixture of dioxane/water (1:2, 300 ml) and Fmoc protected by addition ofNaHCO₃ (12.8 g, 155 mmole) and Fmoc-OSu (26.2 g, 77.6 mmole). Aftercompletion the reaction mixture is extracted three times withdiisopropylether. The product containing aqueous layer is adjusted to pH2 with HCl and the product is extracted with ethyl acetate three times.The combined organic layers are washed with H₂O to neutral pH. The ethylacetate is evaporated and the residual water removed as an azeotropewith acetonitrile. Therefore the product is obtained as a dry foam: 34.9g, 73.7 mmole, 95% of the theory, ESI-MS: monoisotopicM_(W calc).=473.2, M_(W) [M+H]⁺=474.1.

(b) Synthesis of H-Asp(OtBu)-hGlu(OBzI)-D-Val-Arg(Pbf)-Gly-OH

The solid phase peptide synthesis is carried out following the Fmoc/tBustrategy (Atherton E., et. al., J. Chem. Soc., Chem. Comm., 539 (1978)),H-Gly-2-CITrt (46 g, 34.5 mmole) is used as the base resin, coupling isperformed by Fmoc-Xaa-OH/DIC/HOBt (2 eq.:4 eq.:3 eq.) over night, theremoval of the Fmoc protection is achieved by 20% piperidine in DMFafter 5 and 10 min. Alternating washing steps three times withdimethylformamide/isopropanol are employed after each coupling andde-protection step respectively. The amino acid derivatives used intheir chronological order are Fmoc-Arg(Pbf)-OH, Fmoc-D-Val-OH,Fmoc-homoGlu(OBzI)-OH and Fmoc-Asp(OtBu)-OH. The Fmoc-SPPS yields 73.4 gof linear peptide resin (weight gain of the resin 27.4 g, 87% of thetheory, theory=31.5 g).

The side chain protected linear pentapeptide is cleaved from the resin(72.0 g) by a mixture of1,1,1,3,3,3-hexafluoro-2-propanol/dichloromethane 1:4 (700 ml) in threerepetitions. The solvents of the combined filtrates are removed underreduced pressure and the resulting oil stirred in coldmethyl-t-butylether (1 L) to yield an off-white precipitate which isfiltered off, washed three times with methylt-butylether and dried invacuo: 23.6 g, 23.9 mmole, 70% of theory with regard to the loading ofthe base resin, >40 area % on HPLC, retention time of 14.1 min (HPLCconditions: column=Halo® Peptide ES-C18, 4.6×150 mm, 2.7 μm, gradient:linear acetonitrile gradient from 25% to 90% B in 30 min., buffer A=0.1%TFA and 2% acetonitrile in water, buffer B=0.1% TFA in acetonitrile,wavelength=210 nm), ESI-MS: monoisotopic M_(W calc).=986.5, M_(W)[M+H]⁺=987.6.

(c) Synthesis of Cyclo[-Asp(OtBu)-hGlu(OBzI)-D-Val-Arg(Pbf)-Gly-]

The linear side chain protected pentapeptideH-Asp(OtBu)-hGlu(OBzI)-D-Val-Arg(Pbf)-Gly-OH (23.6 g, 23.9 mmole) andthe in-situ activation reagent PyBOP (12.4 g, 23.9 mmol) are dissolvedin 10 L of dimethylformamide (DMF) and added drop wise within 3 h to asolution of additional PyBOP (24.9 g, 47.8 mmole) and Hünig's base (16.4ml, 95.6 mmole) in 5 L DMF. The resulting solution is stirred overnight. The DMF is removed in vacuo and the obtained oil dissolved underreflux in 1.8 L ethanol and crystallized by the addition of 3.2 L ofwater at −18° C. The precipitate is filtered off and washed with waterand ether. In addition it is further purified by silica gelchromatography (150 g silica gel 60, eluent: dichloromethane/methanol9:1) resulting in 3.6 g of the cyclic pentapeptide with a purity >91area % on HPLC (HPLC conditions: column=Halo® Peptide ES-C18, 4.6×150mm, 2.7 μm, gradient: linear acetonitrile gradient from 25% to 90% B in30 min., buffer A=0.1% TFA and 2% acetonitrile in water, buffer B=0.1%TFA in acetonitrile, wavelength=210 nm); 3.7 mmole, 15% of the theory,retention time 15.8 min, ESI-MS: monoisotopic M_(W calc).=968.4, M_(W)[M+H]⁺=969.5.

(d) Synthesis of Cyclo[-Asp(OtBu)-hGlu-D-Val-Arg(Pbf)-Gly-]

The benzyl ester is specifically cleaved by hydrogenolysis. For this,3.6 g (3.7 mmole) of cyclo[-Asp(OtBu)-hGlu(OBzI)-D-Val-Arg(Pbf)-Gly-]are dissolved in 20 ml DMF and diluted with 2 L methanol. After additionof 5 g of 5% palladium on activated charcoal to this solution, themixture is hydrogenated. Upon completion the catalyst is filtered offand the solution concentrated under reduced pressure. The product isprecipitated in methyl-t-butylether to yield 3.0 g of the desiredproduct: 3.4 mmole, yield 93% of the theory, purity: 75 area % on HPLC(HPLC conditions: column=Halo® Peptide ES-C18, 4.6×150 mm, 2.7 μm,gradient: linear acetonitrile gradient from 25% to 90% B in 30 min.,buffer A=0.1% TFA and 2% acetonitrile in water, buffer B=0.1% TFA inacetonitrile, wavelength=210 nm), retention time 13.8 min.

(e) Synthesis of Cyclo[-Asp-hGlu(DMA)-D-Val-Arg-Gly-]

1.5 g (1.7 mmole) of the cyclo pentapeptidecyclo[-Asp(OtBu)-hGlu-D-Val-Arg(Pbf)-Gly-] are conjugated to2,3-dimyristyl-1-amino-sn-glycerol (DMA; 1.0 g, 2.0 mmole) in 100 ml DMFby PyBOP/DIPEA activation (0.9 g, 1.7 mmol/0.6 ml, 3.4 mmole). Thereaction mixture is stirred over night. Then 200 ml dichloromethane areadded and the organic phase is extracted three times with 50 ml 2% KHSO₄and three times with water. The organic layer is evaporated underreduced pressure and the residual water removed as an azeotrope withacetonitrile. The resulting foam is directly treated with the finalcleavage cocktail TFA/H₂O/tri-isopropylsilane/dithioerythritol(92.5:2.5:2.5:2.5) for 1.5 hours and afterwards the solution added dropwise to cold diisopropylether (5° C.) in order to precipitate thedesired product. The residue is then separated by filtration, washedtwice with diisopropylether and in addition dried in vacuo to give 0.5 gof the title compound: 0.5 mmole, 30% of the theory, >93.0 area % onHPLC (HPLC conditions: column=Zorbax SB-C3, 4.6×250 mm, 5 □m, gradient:linear acetonitrile gradient from 30% to 100% B in 25 min., bufferA=0.1% TFA and 2% acetonitrile in water, buffer B=0.1% TFA inacetonitrile, wavelength=210 nm), retention time of 22.0 min, ESI-MS:monoisotopic M_(W calc).=1035.8, M_(W) [M+H]⁺=1037.1.

Example 10: Preparation of pVision-RFP-C Vector Containing, FolateDecorated Liposomes

478.2 mg POPC, 58.8 mg Chol, 13.5 mg folate-lipid (see example 8) and 2μg 7-nitrobenzofurazan-labelled-DOPE are dissolved in 750 μL ethanol(96%) at 60° C. and injected into 4.25 mL of a RFP plasmide containingPBS pH 7.4 (1.27 mg RFP-Plasmid/mL). Molar ratio of the used lipids is77.99:18.83:1.02:0.27. After extrusion through 200 nm polycarbonatemembrane for 5 times and 100 nm polycarbonate membrane for 5 times anddiafiltration the liposomes have an average size of 161 nm with a PDI of0.13. The molar ratio of POPC:Chol is 77.99:15.76, Folate-lipid contentis 502 μg/ml (targeted 770 μg/ml) according to HPLC analysis.

Example 11: Preparation of Anis Amide Decorated Liposomes

470 mg POPC, 60 mg Chol and 13.5 mg anis amide lipid (see example 4) aredissolved in 750 μL ethanol (96%) at 55° C. and injected into 4.25 mL ofPBS pH 7.4. Molar ratio of the used lipids is 77.99:18.83:1.02:0.27.After extrusion through 100 nm polycarbonate membrane the liposomes havean average size of 110 nm with a PDI of 0.068. According to HPLCanalysis the anis amide lipid content is 72% of the theoretical value.

Example 12: Preparation of RGD Decorated Liposomes

A mixture of DOPC, Chol, NBD-DOPE, and the RGD-lipid obtained in Example9 in a molar ratio of DOPC:Chol:NBD-DOPE:RGD-lipid 66:33:0.5:0˜5 areused to prepare liposomes by dry film method in HEPES buffer, followedby extrusion through 200 nm polycarbonate membrane for 5 times and 100nm polycarbonate membrane for 21 times using Lipofast extruder (Avestin,Inc., Ottawa, Canada). The obtained liposomes are stored at 4° C. untiluse.

Example 13: Cellular Uptake of RGD Decorated Liposomes

The extent of cellular uptake for RGD decorated liposomes (obtained inExample 6) on M21 cells are evaluated on the basis of NBD-DOPE signaldetected by Guava easyCyte 8HT flowcytometer and is illustrated in FIG.1 and Table 1.

TABLE 1 NBD positive cells (%) Avg SD Blank 0.19 0.36 0.4 0.33 0.29 0.650.37 0.15 0% 1.04 1.75 1.56 1.57 1.37 1.66 1.49 0.25 RGD 5% 29.99 28.4123.77 24.82 23.68 20.99 25.28 3.33 RGD

About a 16 fold enhancement in cellular uptake is observed for the RGDtargeting liposome (5% DMA-RGD) as compared to non-targeting liposome(0% DMA-RGD). The x-axis represents the molar ratio of DMA-RGD (%) inthe liposome. The y-axis represents NBD-positive cells (%). FIG. 1illustrates that the RGD moieties can recognize target receptors(Integrin α_(v)β₃ receptors) expressed on M21 cells (* p<0.01).

Example 14: Synthesis of(5S,8S,45S,E)-11-(2-amino-2-oxoethyl)-45-(3-((2,3-bis(tetradecyloxy)propyl)amino)-3-oxopropyl)-5,8-dimethyl-3,6,9,12,15,43-hexaoxo-1-phenyl-2,19,22,25,28,31,34,37,40-nonaoxa-4,7,10,11,16,44-hexaazahexatetracont-13-en-46-oicacid

(a) Synthesis of Fmoc-Glu(DMA)-Diphenylmethyl Resin (See Example 7, 1.1Eq., 3.05 Mmol) (b) Synthesis ofRR11a-NH-PEG₈-PA-Glu(DMA)-diphenylmethyl resin

RR11a-NH-PEG₈-PA-Glu(DMA)-diphenylmethyl resin is obtained throughconventional solid phase synthesis by the following reaction sequence:

(1) cleavage of the Fmoc group of the Fmoc-Glu(DMA)-diphenylmethyl resinwith piperidin in DMF,

(2) condensation with Fmoc-NH-PEG₈-PA using PyBOP in DMF and DIPEA,

(3) cleavage of the Fmoc group of theFmoc-NH-PEG₈-PA-Glu(DMA)-diphenylmethyl resin with piperidin in DMF andfinally

(4) condensation with RR11a-OH using PyBOP in DMF and DIPEA.

(c) Synthesis of(5S,8S,45S,E)-11-(2-amino-2-oxoethyl)-45-(3-((2,3-bis(tetradecyloxy)propyl)amino)-3-oxopropyl)-5,8-dimethyl-3,6,9,12,15,43-hexaoxo-1-phenyl-2,19,22,25,28,31,34,37,40-nonaoxa-4,7,10,11,16,44-hexaazahexatetracont-13-en-46-oicacid

7.15 g RR11a-NH-PEG₈-PA-Glu(DMA)-diphenylmethyl resin are washed with 50ml dichloromethane each, filtered off, suspended again in 50 mldichloromethane and dried in vacuo. Then 70 ml of a 5% ic solution oftrifluaroacetic acid in dichloromethane were added. The suspension isstirred at room temperature for 3.5 hour and then filtered into 100 mlcold diisopropylether. The resin is rinsed withdichloromethane/diisopropylether (1/1). The combined filtrates areevaporated in vacuo and lyophilyzed from t-BuOH to yield 4.15 g (92%) ofan amber solid. ESI-MS: monoisotopic M_(W calc).=1481.9, M_(W)[M−H]⁻=1480.2.

Example 15: Synthesis of(5S,8S,45S,E)-11-(2-amino-2-oxoethyl)-45-(3-((2,3-bis(tetradecyloxy)propyl)amino)-3-oxopropyl)-5,8-dimethyl-3,6,9,12,15,43-hexaoxo-1-phenyl-2,19,22,25,28,31,34,37,40-nonaoxa-4,7,10,11,16,44-hexaazahexatetracont-13-en-46-oicacid

7.15 g RR11a-NH-PEG₈-PA-Glu(DMA)-OH (product of Example 14) and 1.50 mlDIPEA are dissolved in 70 ml dichloromethane. Then 4.32 g MeO-PEG-NH2and 1.67 g PyBOP are added and the solution is stirred overnight. Thebrown solution is evaporated and the residue is purified twice by columnchromatography over 300 g silica gel (Merck 60, 0.040-0.063 mm) using amixture of ethylacetat, methanol and triethylamine in a ratio of 16:3.1resp. 17:2:1. The product containing fractions are combined andevaporated and the resulting viscous residue is lyophilized from t-BuOHto yield 4.5 g (60%) of an yellowish solid. MALDI-MS: monoisotopicM_(W calc).=3476.2, M_(W) [M+Na]+=3500, M_(n)=3363.2, M_(W)=3384.5,PDI=1.01

Example 16: Synthesis of benzyl((2S,5S,145,E)-8-(2-amino-2-oxoethyl)-14-carbamoyl-5-methyl-3,6,9,12,17-pentaoxo-20-(tetradecyloxy)-22-oxa-4,7,8,13,18-pentaazahexatriacont-10-en-2-yl)carbamate

(a) Synthesis of Fmoc-Glu(DMA)-Sieber Resin

In a 100 ml SPPS reactor 5.0 g of Sieber resin (3.1 mmol) are washedtwice with 50 ml DMF, treated with a 20% ic solution of piperidine inDMF over 15 min and washed three times alternatingly with 50 ml DMF andwith 50 ml iPrOH. Then a solution of 3.2 g of(2S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-glutamicacid-γ-2,3-bis(tetradecyloxy)propyl-amide (see example 2, 1.25 eq., 3.8mmol) and 2.48 g PyBOP (1.5 equ.) in 50 ml DMF, and 1.62 ml DIPEA (2.5equ.) for 2.5 h. The solution is filtered off and the resin is washedthree times alternatingly with 50 ml DMF and with 50 ml iPrOH.

(b) Synthesis of RR11a-Glu(DMA)-Sieber Resin

RR11a-Glu(DMA)-Sieber resin is obtained through conventional solid phasesynthesis by the following reaction sequence:

(1) cleavage of the Fmoc group of the Fmoc-Glu(DMA)-Sieber resin withpiperidin in DMF (5.6 g resin after drying in vacuo).

(2) condensation with RR11a-NHS using DIPEA in DMF.

(c) Cleavage of the Product from the Resin:

2.6 g RR11a-Glu(DMA)-Sieber resin are treated with 20 ml 5% ictrifluaroacetic acid in dichloromethane for 2 h. The suspension isfiltered into 100 ml cold diisopropylether. The filtrate is evaporatedin vacuo and lyophilyzed from t-BuOH to yield 660 mg of a yellowishsolid. ESI-MS: monoisotopic M_(W calc).=1056.7, M_(W) [M−H]⁻=1056.0.

Example 17: Synthesis of benzyl((2S,5S,42S,E)-8-(2-amino-2-oxoethyl)-42-carbamoyl-5-methyl-3,6,9,12,40,45-hexaoxo-48-(tetradecyloxy)-16,19,22,25,28,31,34,37,50-nonaoxa-4,7,8,13,41,46-hexaazatetrahexacont-10-en-2-yl)carbamate

(a) Synthesis of Fmoc-Glu(DMA)-Sieber Resin: (See Example 16) (b)Synthesis of NH₂-PEG₈-PA-Glu(DMA)-Sieber Resin

NH₂-PEG₈-PA-Glu(DMA)-Sieber resin is obtained through conventional solidphase synthesis by the following reaction sequence:

(1) cleavage of the Fmoc group of the Fmoc-Glu(DMA)-Sieber resin withpiperidin in DMF,

(2) condensation with Fmoc-NH-PEG₈-PA using HBTU in DMF and DIPEA andfinally

(3) cleavage of the Fmoc group of the Fmoc-NH-PEG₈-PA-Glu(DMA)-Sieberresin with piperidin in DMF.

(c) Synthesis of NH₂-PEG₈-PA-Glu(DMA)-Amide

The product is cleaved from the NH₂-PEG₈-PA-Glu(DMA)-Sieber resin usingtrifluaroacetic acid in dichloromethane. ESI-MS: monoisotopicM_(W calc).=1034.8, M_(W) [M+H]⁺=1035.9.

(d) Synthesis of benzyl((2S,5S,42S,E)-8-(2-amino-2-oxoethyl)-42-carbamoyl-5-methyl-3,6,9,12,40,45-hexaoxo-48-(tetradecyloxy)-16,19,22,25,28,31,34,37,50-nonaoxa-4,7,8,13,41,46-hexaazatetrahexacont-10-en-2-yl)carbamate

A 5 ml round bottom flask equipped with mechanical stirrer is chargedwith 42 mg of NH₂-PEG8-PA-Glu(DMA)-amide (40.6 mmol) in 2 mldichloromethane. Then 0.01 ml triethylamine (95 mmol) are added. A lightyellow solution results after 2-3 minutes of stirring and 23 mg ofRR11a-NHS (41 mmol) are added over a period of 3 min. The solution isstirred for 1 hr and evaporated under reduced pressure resulting in aoff-white solid product. The product shows a single spot in TLC.M_(W calc).=1480.0, M_(W) [M+H]⁺=1482 and M_(W) [M+Na]⁺=1504.0.

Example 18: Synthesis of benzyl((2S,5S,126S,E)-8-(2-amino-2-oxoethyl)-126-carbamoyl-5-methyl-3,6,9,12,124,129-hexaoxo-132-(tetradecyloxy)-16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,79,82,85,88,91,94,97,100,103,106,109,112,115,118,121,134-heptatriacontaoxa-4,7,8,13,125,130-hexaazaoctatetracontahect-10-en-2-yl)carbamate

(a) Synthesis of Fmoc-Glu(DMA)-Sieber Resin: (See Example 16) (b)Synthesis of RR11a-NH-PEG₃₆-PA-Glu(DMA)-Sieber Resin

RR11a-NH-PEG₃₆-PA-Glu(DMA)-Sieber resin is obtained through conventionalsolid phase synthesis by the following reaction sequence:

(1) cleavage of the Fmoc group of the Fmoc-Glu(DMA)-Sieber resin withpiperidin in DMF,

(2) condensation with Fmoc-NH-PEG₃₆-PA using PyBOP in DMF and DIPEA,

(3) cleavage of the Fmoc group of the Fmoc-NH-PEG₃₆-PA-Glu(DMA)-Sieberresin with piperidin in DMF and finally

(4) condensation with RR11a-NHS using DIPEA in DMF.

(c) Synthesis of benzyl((2S,5S,126S,E)-8-(2-amino-2-oxoethyl)-126-carbamoyl-5-methyl-3,6,9,12,124,129-hexaoxo-132-(tetradecyloxy)-16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76,79,82,85,88,91,94,97,100,103,106,109,112,115,118,121,134-heptatriacontaoxa-4,7,8,13,125,130-hexaazaoctatetracontahect-10-en-2-yl)carbamate

7.0 g RR11a-NH-PEG₃₆-PA-Glu(DMA)-Sieber resin are treated with 70 ml ofa 2% ic solution of trifluaroacetic acid in dichloromethane are added.The suspension is stirred at room temperature for 3 h and then filteredinto 70 ml cold diisopropylether. The filtrate is evaporated in vacuoand lyophilyzed from t-BuOH to yield 1.25 g of a white solid. ESI-MS:monoisotopic M_(W calc).=2713.7, M_(W) [M+Na+H]²+=1380.1.

Example 19: Preparation of RR11a Decorated Liposomes

RR11a decorated liposomes (MS 15-4) and control liposomes (MS 15-0) arecomposed from the following lipid solutions:

concentration in volume lipid chloroform MS 15-0 MS 15-4 DOPE 33 mM 35μl 35 μl DSPC 32 mM 35 μl 35 μl Cholesterol 33 mM 35 μl 35 μlMPEG2000-DOPE 18 mM 15 μl 15 μl RhB-DOPE 0.8 mM  3 μl  3 μlRR-11a-8PEG-PA- 17 mM  0 μl 20 μl Glu(DMA)-amide(see Example 17)

A 3 ml screw cap glass vial (Teflon lined cap) is charged with the abovelipids and vortexed briefly. The chloroform is evaporated under a streamof Argon until a opaque film of the lipids is obtained. Then the vial isplaced in a desiccator under vacuum for 10 minutes. To the dry film isadded 1000 uL of DPBS 1× and the content is vortexed until a homogenousmilky suspension is obtained (3-4 min). This is followed by bathsonication in a Branson 1510 model for 5 minutes to obtain a cloudysuspension. This suspension is then probe sonicated in a Branson Model4C15 at a 30% of full amplitude for 30 seconds (avoiding foaming) toobtain a nearly translucent suspension of liposomes. The suspension ishigh pressure extruded though 100 nm polycarbonate membrane (Avanti No610005) Finally the suspension is steril filtered though 0.22 μmMillex-GV membrane filters and stored in a steril vial at 4° C. The Zavg. hydrodynamic diameter of MS-15-0 and 15-4 is 101 and 99 um (MalvernZetaSizer instrument), respectively.

Example 20: Legumain Targeting of RR11a Decorated Liposomes

Legumain targeting experiments are performed according to the followingprotocol employing the liposomal formulations of Example 19:

Day 1 seed 3.12×10e4 4T1 cells/cm2 on untreated glass slide

Day 2 add 100 uM CoCl2; incubate 24 h

Day 3 add 100 ul liposomes (10e12 NPs/ml); incubate 2 h;

add 5 ug/ml Hoechst 33342; incubate 20 min; mount and analyze with afluorescent microscope

Cell culture medium: RPMI 1640 1× with L-glutamine, supplemented with10% FBS, 10 mM Hepes, 0.075% w/v sodium bicarbonate and 1 mM sodiumpyruvate.

Pictures are acquired from random fields from each portaobjects, using63× objective, 2×2 bin, 500 ms for Hoechst, 1000 ms for RhodB and 100 msclear field, and 20-30 images stack with 0.5 um height focus step aroundnucleus focus point for image deconvolution analysis. FIG. 2 respresentsone of the 20-30 images from the stack, after deconvolution, showing anintermediate focus point. Clearly the data show colocalization ofRhB-DOPE with cells and therefore demonstrate that liposomes made withRR-11a-8PEG-PA-Glu(DMA)-amide target these cells effectively compared tothe non-targeted control.

Example 21: Synthesis of Antibody (Fc Unit) Targeting Lipid, DisulfideBridged DecapentapeptideH-Glu(DMA)-Ala-Asp-Cys-Ala-Trp-his-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-OH

(a) Synthesis of LinearH-Glu(DMA)-Ala-Asp-Cys-Ala-Trp-his-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-OH

The solid phase peptide synthesis is carried out with solid phasesynthesizer ABI 431A following the Fmoc/tBu strategy (Atherton E., et.al., J. Chem. Soc., Chem. Comm., 539 (1978)), H-Thr(tBu)-2-CITrt (0.5 g,0.25 mmole) is used as the base resin. The amino acid derivatives usedin their chronological order are Fmoc-Cys(Trt)-OH, Fmoc-Trp(Boc)-OH,Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH,Fmoc-His(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Ala-OH and Fmoc-Glu(DMA)-OH. Coupling isperformed by Fmoc-Xaa-OH/Trimethylpyridine/HBTU (4 eq.:4 eq.:3.6 eq.),except Fmoc-Glu(DMA)-OH which is coupled using DIPEA/PyBOP (4.8 eq.:7.2eq.:4.8 eq.). The removal of the Fmoc protection is achieved by 20%piperidine in DMF. Alternating washing steps three times withdimethylformamide are employed after each coupling and de-protectionstep respectively. The Fmoc-SPPS yields 1.4 g of linear peptide resin.

The linear decapentapeptide is cleaved from the resin by a mixture oftrifluoroaceti acid/triisopropylsilane/dithioerythritol/water(92.5:2.5:2.5:2.5, 14 ml) during 2.5 hours. After filtration thefiltrate is diluted with 140 ml diisopropylether. The brownish solid isfiltered off and dried in vacuo: 485 mg, 37% of theory, ESI-MS:monoisotopic M_(W calc).=2198.2, M_(W) [M+2H]²⁺=1099.6.

(b) Synthesis of Disulfide BridgedH-Glu(DMA)-Ala-Asp-Cys-Ala-Trp-his-Leu-Gly-Glu-Leu-Val-Trp-Cys-Thr-OH

10 mg of the brownish solid obtained under Example 21 a) are dissolvedin 10 ml methanol and brought to pH 8 by the addition of DIPEA. Thesolution is stirred under oxygen atmosphere over night. Evaporation ofthe solution provids the final product as a brownish solid inquantitative yield. ESI-MS: monoisotopic M_(W calc).=2196.2, M_(W)[M+2H]²⁺=1098.6.

Example 22: Preparation of RR11a Decorated Liposomes without Extrusion

RR11a decorated liposomes (MS-32-1 to MS-32-10) are composed from thefollowing lipid solutions:

A 3 ml screw cap glass vial (Teflon lined cap) is charged with the abovelipids and vortexed briefly. The chloroform is evaporated under a streamof Argon until a opaque film of the lipids is obtained. Then the vial isplaced in a desiccator under vacuum for 10 minutes. To the dry film isadded 1000 uL of DPBS 1× and the content is vortexed until a homogenousmilky suspension is obtained (10 min). This is followed by bathsonication in a Branson 1510 model for 5 minutes to obtain a cloudysuspension. This suspension is then probe sonicated in a Branson Model4C15 at a 40% of full amplitude for 30 seconds (avoiding foaming) toobtain a nearly translucent suspension of liposomes. Finally thesuspension is steril filtered though 0.22 μm Millex-GV membrane filtersand stored in a steril vial at 4° C. The Z avg. hydrodynamic diameterand PDI are determined using a Malvern ZetaSizer instrument:

formulation MS- MS- concentration in MS-32-1 MS-32-2 MS-32-3 MS-32-4MS-32-5 MS-32-6 MS-32-7 MS-32-8 32-9 32-10 lipid chloroform [mM] Volume[μl] DOPE 33 35 DSPC 33 35 Cholesterol 33 35 RhB-DOPE 0.8 3 MPEG2000- 18— — 15 15 — — 15 15 — — DOPE RR-11a- 24 — — — — 15 — 15 — — —Glu(DMA)-amide (see Example 16) RR-11a-8PEG- 17 15 — 15 — — — — — — —PA-Glu(DMA)- amide RR-11a-36PEG- 9 — — — — — 15 — 15 35 70 PA-Glu(DMA)-amide RR-11a-8PEG- 9 — 15 — 15 — — — — — — PA-Glu(DMA)- NH-MPEG2k (seeExample 15) Z avg. hydrodynamic 204.9 115.1 109.0 98.9 179.8 144.6 227.0100.6 — — diameter PDI 0.5 0.2 0.2 0.19 0.25 0.28 0.47 0.18 — —

The invention claimed is:
 1. A carrier system comprising a compound offormula I

wherein Y represents O, N, S or a covalent bond, S₁, S₂, S₃ representindependently of each other a covalent bond or a spacer group, X₁, X₂,X₃ represent independently of each other H or a ligand group or any twoof X₁, X₂, X₃ may together form a ligand group, wherein at least one ofX₁, X₂, X₃ is a legumain targeting ligand group, L is a group of formula(a)

wherein the dashed line represents the linkage to N, R₁ represents H ora group of formula —(CH₂)₂—OR_(b1), R₁′ represents H or a group offormula —(CH₂)₂—OR_(b2), R₂ represents H or a group of formula—CH₂—OR_(c), R₂′ represents H or a group of formula —OR_(d) or—CH₂—OR_(d), R₃ represents H or a group of formula —(CH₂)₂—OR_(e) or—(CH₂)₃—OR_(e), R_(a), R_(b1), R_(b2), R_(c), R_(d), R_(e) representindependently of each other a saturated or unsaturated, straight orbranched hydrocarbon chain, m is 1, 2 or 3, with the proviso that atleast one of R₁, R₁′, R₂, R₂′, R₃ is not H, and wherein at least one ofthe following conditions is satisfied: the carrier system comprises apharmaceutically active agent in a solvated form; or the carrier systemcomprises one or more pteroyl derivatives and/or aza-peptidederivatives; or the carrier system has been lyophilized or freeze-driedand therefore has enhanced stability; or wherein at least one of X₁, X₂,X₃ is a ligand that is capable of binding to an antibody, antibodyfragment, engineered antibody or an antibody mimic.
 2. The carriersystem according to claim 1, which comprises a pharmaceutically activeagent in a solvated form.
 3. The carrier system according to claim 1,which comprises one or more pteroyl derivatives and/or aza-peptidederivatives.
 4. The carrier system according to claim 1, which has beenlyophilized or freeze-dried and therefore has enhanced stability.
 5. Thecarrier system according to claim 1, wherein at least one of X₁, X₂, X₃is a ligand that is capable of binding to an antibody, antibodyfragment, engineered antibody or an antibody mimic.
 6. The carriersystem according to claim 1, wherein R₃ is H, and L is a group offormulas (b) or (c)

wherein the dashed line represents the linkage to N, and S₁, S₂, S₃, X₁,X₂, X₃, Y, R_(a), and m are defined as for formula I, with the provisothat in formula (b) one of R₂ and R₂′ is not H, and in formula (c) oneof R₁ and R₁′ is not H, and at least one of X₁, X₂, X₃ is a legumaintargeting ligand group.
 7. The carrier system according to claim 6,wherein L is a group of formula (b1), (b2), (b3) or (b4):

wherein the dashed line represents the linkage to N, and wherein R_(a),R_(c) and R_(d) are independently of each other a saturated orunsaturated, straight or branched hydrocarbon chain.
 8. The carriersystem according to claim 6, wherein L is a group of formula (c1) or(c2):

wherein the dashed line represents the linkage to N, and wherein R_(a),R_(b1), R_(b2) are independently of each other a saturated orunsaturated, straight or branched hydrocarbon chain.
 9. The carriersystem according to claim 1, wherein R₁, R₁′, R₂, R₂′ are H, R₃ is agroup of formula —(CH₂)₂—OR_(e) or —(CH₂)₃—OR_(e), and S₁, S₂, S₃, X₁,X₂, X₃, Y, R_(a), and m are defined as for formula I.
 10. The carriersystem according to claim 1, wherein R_(a), R_(b1), R_(b2), R_(c),R_(d), R_(e) are independently of each other straight or branchedC(10-22)alkyl, C(10-22)alkenyl or C(10-22)alkynyl.
 11. The carriersystem according to claim 10, wherein C(10-22)alkenyl andC(10-22)alkynyl have 1, 2, 3 or 4 unsaturated bonds.
 12. The carriersystem according to claim 10, wherein C(10-22)alkenyl andC(10-22)alkynyl have 1 or 2 unsaturated bonds.
 13. The carrier systemaccording to claim 1, wherein the carrier system is a microparticulateor a nanoparticulate material.
 14. The carrier system according to claim13, wherein the microparticulate or a nanoparticulate material is aliposome or a micelle, comprising at least one compound of formula I andoptionally one or more other co-lipids.
 15. The carrier system accordingto claim 13, wherein the microparticulate or a nanoparticulate materialis a lipid vesicle, a nanoparticle, a nanosphere and/or a nanorod,comprising at least one compound of formula I and optionally one or moreother co-lipids.
 16. The carrier system according to claim 15, whereinthe lipid vesicle further contains at least one bioactive agent enclosedor embedded within its internal void or adsorbed onto or attached to itssurface.
 17. The carrier system according to claim 1, wherein the spacergroup is polyethylene glycol or an end-capped polyethylene glycol. 18.The carrier system according to claim 1, wherein the at least one of X₁,X₂, X₃ that is a legumain targeting ligand group is RR11a.
 19. Thecarrier system according to claim 1, wherein the carrier system is aliposome, and the at least one of X₁, X₂, X₃ that is a legumaintargeting ligand group is RR11a.
 20. The carrier system according toclaim 1, wherein, in addition to at least one of X₁, X₂, X₃ being alegumain targeting ligand group, at least one further of X₁, X₂, X₃ ortwo of X₁, X₂, X₃ together is a targeting ligand or an antigenic ligandor a therapeutic or diagnostic ligand or a combination thereof.
 21. Apharmaceutical composition comprising the carrier system according toclaim 1 and a pharmaceutically acceptable carrier.
 22. A drug deliverysystem, diagnostic system or as an antigen display system, said systemcomprising the carrier system according to claim 1 and apharmaceutically acceptable carrier.
 23. A method for the diagnosis of adisease by a disease specific diagnostic agent containing a legumaintargeting ligand group, comprising administering to a host in needthereof a carrier system comprising a compound of formula I

wherein Y represents O, N, S or a covalent bond, S₁, S₂, S₃ representindependently of each other a covalent bond or a spacer group, X₁, X₂,X₃ represent independently of each other H or a ligand group or any twoof X₁, X₂, X₃ may together form a ligand group, wherein at least one ofX₁, X₂, X₃ is a legumain targeting ligand group, L is a group of formula(a)

wherein the dashed line represents the linkage to N, R₁ represents H ora group of formula —(CH₂)₂—OR_(b1), R_(1′) represents H or a group offormula —(CH₂)₂—OR_(b2), R₂ represents H or a group of formula—CH₂—OR_(c), R_(2′) represents H or a group of formula —OR_(d) or—CH₂—OR_(d), R₃ represents H or a group of formula —(CH₂)₂—OR_(e) or—(CH₂)₃—OR_(e), R_(a), R_(b1), R_(b2), R_(c), R_(d), R_(e) representindependently of each other a saturated or unsaturated, straight orbranched hydrocarbon chain, m is 1, 2 or 3, with the proviso that atleast one of R₁, R_(1′), R₂, R_(2′), R₃ is not H, wherein at least oneof Xi, X₂, X₃ is said diagnostic agent containing a legumain targetingligand group; or a method for modulating an immune response, comprisingadministering to a host in need thereof the carrier system comprisingthe compound of formula I, wherein at least one of X₁, X₂, X₃ is anantigenic agent containing a legumain targeting ligand group; or amethod for treating cancer, comprising administering to a subject inneed thereof an effective amount of the carrier system comprising thecompound of formula I, wherein said carrier system comprises a cancertherapeutic agent; or a method for treating an inflammatory disease,comprising administering to a subject in need thereof an effectiveamount of the carrier system comprising the compound of formula I,wherein said carrier system comprises an anti-inflammatory therapeuticagent; or a method for transporting a biologically active compoundacross a membrane and/or a method for delivering a biologically activecompound into a cell, comprising achieving said transporting or deliveryby the carrier system comprising the compound of formula I, whichcomprises said biologically active compound; or a method for eliciting apsychological response from a human patient, comprising administering tosaid human the carrier system comprising the compound of formula I,which comprises one or more bioactive agents; or a method for preventinga psychotic episode in a human subject, comprising administering to saidhuman the carrier system comprising the compound of formula I, whichcomprises one or more antipsychotic agents; or a method for in vitroapplication for growth promotion and differentiation of cells ormodification of expression profiles and post-translational modificationpatterns of biological products manufactured in a bioreactor, comprisingadministering into said bioreactor the carrier system comprising thecompound of formula I, which bioreactor further contains said cells orbiological products; or a method for preventing a disease in a humansubject, comprising administering to said human the carrier systemcomprising the compound of formula I, which comprises one or morevaccines.
 24. The method according to claim 23, which is for thediagnosis of a disease by a disease specific diagnostic agent containinga legumain targeting ligand group, comprising administering to a host inneed thereof the carrier system comprising the compound of formula I,wherein at least one of X₁, X₂, X₃ is said diagnostic agent containing alegumain targeting ligand group.
 25. The method according to claim 23,which is for modulating an immune response, comprising administering toa host in need thereof the carrier system comprising the compound offormula I, wherein at least one of X₁, X₂, X₃ is an antigenic agentcontaining a legumain targeting ligand group.
 26. The method accordingto claim 23, which is for treating cancer, comprising administering to asubject in need thereof an effective amount of the carrier systemcomprising the compound of formula I, wherein said carrier systemcomprises a cancer therapeutic agent.
 27. The method according to claim23, which is for treating an inflammatory disease, comprisingadministering to a subject in need thereof an effective amount of thecarrier system comprising the compound of formula I, wherein saidcarrier system comprises an anti-inflammatory therapeutic agent.
 28. Themethod according to claim 23, which is for transporting a biologicallyactive compound across a membrane and/or a method for delivering abiologically active compound into a cell, comprising achieving saidtransporting or delivery by the carrier system comprising the compoundof formula I, which comprises said biologically active compound.
 29. Themethod according to claim 23, which is for eliciting a psychologicalresponse from a human patient, comprising administering to said humanthe carrier system comprising the compound of formula I, which comprisesone or more bioactive agents.
 30. The method according to claim 23,which is for preventing a psychotic episode in a human subject,comprising administering to said human the carrier system comprising thecompound of formula I, which comprises one or more antipsychotic agents.31. The method according to claim 23, which is for in vitro applicationfor growth promotion and differentiation of cells or modification ofexpression profiles and post-translational modification patterns ofbiological products manufactured in a bioreactor, comprisingadministering into said bioreactor the carrier system comprising thecompound of formula I, which bioreactor further contains said cells orbiological products.
 32. The method according to claim 23, which is forpreventing a disease in a human subject, comprising administering tosaid human the carrier system comprising the compound of formula I,which comprises one or more vaccines.
 33. The method according to claim23, wherein, in the compound of formula I, R₃ is H, and L is a group offormulas (b) or (c)

wherein the dashed line represents the linkage to N, and S₁, S₂, S₃, X₁,X₂, X₃, Y, R_(a), and m are defined as for formula I, with the provisothat in formula (b) one of R₂ and R₂′ is not H, and in formula (c) oneof R₁ and R₁′ is not H, and at least one of X₁, X₂, X₃ is a legumaintargeting ligand group.
 34. The method according to claim 23, wherein,in the compound of formula I, L is a group of formula (b1), (b2), (b3)or (b4):

wherein the dashed line represents the linkage to N, and wherein R_(a),R_(c) and R_(d) are independently of each other a saturated orunsaturated, straight or branched hydrocarbon chain; or L is a group offormula (c1) or (c2):

wherein the dashed line represents the linkage to N, and wherein R_(a),R_(b1), R_(b2) are independently of each other a saturated orunsaturated, straight or branched hydrocarbon chain.
 35. The methodaccording to claim 23, wherein, in the compound of formula I, R₁, R₁′,R₂, R₂′ are H, R₃ is a group of formula —(CH₂)₂—OR_(e) or—(CH₂)₃—OR_(e), and S₁, S₂, S₃, X₁, X₂, X₃, Y, R_(a), and m are definedas for formula I; and/or R_(a), R_(b1), R_(b2), R_(c), R_(d), R_(e) areindependently of each other straight or branched C(10-22)alkyl,C(10-22)alkenyl or C(10-22)alkynyl.
 36. The method according to claim23, wherein the carrier system has been lyophilized or freeze-dried andtherefore has enhanced stability.